Compositions and methods for eliminating undesired subpopulations of T cells in patients with immunological defects related to autoimmunity and organ or hematopoietic stem cell transplantation

ABSTRACT

The present invention relates generally to methods for stimulating T cells, and more particularly, to methods to eliminate undesired (e.g. autoreactive, alloreactive, pathogenic) subpopulations of T cells from a mixed population of T cells, thereby restoring the normal immune repertoire of said T cells. The present invention also relates to compositions of cells, including stimulated T cells having restored immune repertoire and uses thereof.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to methods for stimulating Tcells to restore normal immune repertoire. The present disclosureincludes methods to eliminate undesired (e.g. autoreactive,alloreactive, pathogenic) subpopulations of T cells from a mixedpopulation of T cells, thereby restoring the normal immune repertoire ofsaid T cells. The present invention also relates to compositions ofcells, including stimulated T cells having restored immune repertoireand uses thereof.

2. Description of the Related Art

The ability of T cells to recognize the universe of antigens associatedwith various cancers or infectious organisms is conferred by its T cellantigen receptor (TCR), which is made up of both an α (alpha) chain anda β (beta) chain or a γ (gamma) and a δ (delta) chain. The proteinswhich make up these chains are encoded by DNA, which employs a uniquemechanism for generating the tremendous diversity of the TCR. Thismultisubunit immune recognition receptor associates with the CD3 complexand binds to peptides presented by the major histocompatibility complex(MHC) class I and II proteins on the surface of antigen-presenting cells(APCs). Binding of TCR to the antigenic peptide on the APC is thecentral event in T cell activation, which occurs at an immunologicalsynapse at the point of contact between the T cell and the APC.

To sustain T cell activation, T lymphocytes typically require a secondco-stimulatory signal. Co-stimulation is typically necessary for a Thelper cell to produce sufficient cytokine levels that induce clonalexpansion. Bretscher, Immunol. Today 13:74, 1992; June et al., Immunol.Today 15:321, 1994. The major co-stimulatory signal occurs when a memberof the B7 family ligands (CD80 (B7.1) or CD86 (B7.2)) on an activatedantigen-presenting cell (APC) binds to CD28 on a T cell.

Methods of stimulating the expansion of certain subsets of T cells havethe potential to generate a variety of T cell compositions useful inimmunotherapy. Successful immunotherapy can be aided by increasing thereactivity and quantity of T cells by efficient stimulation.Furthermore, in the settings of autoimmunity or transplantation,successful immunotherapy can be aided by the elimination of unwantedautoreactive or alloreactive cells.

The various techniques available for expanding human T cells have reliedprimarily on the use of accessory cells and/or exogenous growth factors,such as interleukin-2 (IL-2). IL-2 has been used together with ananti-CD3 antibody to stimulate T cell proliferation, predominantlyexpanding the CD8⁺ subpopulation of T cells. Both the APC signalsdirected towards the TCR/CD3 complex and CD28 on the surface of T cellsare thought to be required for optimal T cell activation, expansion, andlong-term survival of the T cells upon re-infusion. The requirement forMHC-matched APCs as accessory cells presents a significant problem forlong-term culture systems because APCs are relatively short-lived.Therefore, in a long-term culture system, APCs must be continuallyobtained from a source and replenished. The necessity for a renewablesupply of accessory cells is problematic for treatment ofimmunodeficiencies in which accessory cells are affected. In addition,when treating viral infection, if accessory cells carry the virus, thecells may contaminate the entire T cell population during long-termculture.

In the absence of exogenous growth factors or accessory cells, aco-stimulatory signal may be delivered to a T cell population, forexample, by exposing the cells to a CD3 ligand and a CD28 ligandattached to a solid phase surface, such as a bead. See C. June, et al.(U.S. Pat. No. 5,858,358); C. June et al. WO 99/953823. While thesemethods are capable of achieving therapeutically useful T cellpopulations, increased robustness and ease of T cell preparation remainless than ideal.

Methods previously available in the art have made use of anti-CD3 andanti CD28 for the expansion of T cells. In addition, the methodscurrently available in the art have not focused on short-term expansionof T cells or obtaining a more robust population of T cells and thebeneficial results thereof. None of these methods has described usingsuch or similar methods to eliminate an undesired clonal or oligoclonalT cell population from a T cell population nor the beneficial resultsthereof. Moreover, the methods previously available tend to further skewthe clonality of the T cell population rather than eliminate undesiredreactive clones from a T cell population, and restore a normal immunerepertoire. For maximum in vivo effectiveness, theoretically, an exvivo- or in vivo-generated, activated T cell population should be in astate that can maximally orchestrate an immune response to cancer,infectious disease, or other disease states. In the setting ofautoimmunity or transplantation, the activated T cell populations shouldbe in a state to reconstitute a normal T cell repertoire with a reducedpresence or entirely without the presence of autoreactive or potentiallypathogenic alloreactive T cells. Currently, patients with autoimmunediseases are treated with long-term immunosuppression to inhibit theautoreactive T cells that cause disease. When the immunosuppressiveagents are stopped, disease recurs often concomitant with reappearanceof disease causing T cells that re-emerge in these patients. The majorproblem in hematopoietic stem cell transplantation is graft-versus-hostdisease (GVHD), which is caused by alloreactive T cells present in theinfused hematopoietic stem cell preparation. In organ transplantation,graft rejection mediated by alloreactive host T cells is the majorproblem, usually overcome by long-term immunosuppression of thetransplant recipient.

The present invention provides methods to generate an increased numberof more highly activated and more pure T cells that have surfacereceptor and cytokine production characteristics that appear morehealthy and natural than other expansion methods and further providesfor the diminution or elimination of undesired autoreactive oralloreactive populations of T cells. The present invention providesmethods for the use of said populations of T cells in the setting ofautoimmune diseases, hematopoietic stem cell, and organ transplantation,as well as other settings where reconstitution of an ablated, abrogated,or otherwise dysfunctional T cell immune system is desired. In addition,the present invention provides compositions of cell populations of anytarget cell, including T cell populations and parameters for producingthe same, as well as providing other related advantages.

Additionally, it is becoming well recognized that the aging immunesystem is characterized by a progressive decline in the responsivenessto exogenous antigens and tumors in combination with a paradoxicalincrease in autoimmunity (C. Weyand et al. Mechanisms of Ageing andDevelopment 102:131-147, 1998; D. Schmidt et al. Molecular Medicine2:608-618, 1996; G. Liuzzo et al. Circulation 100:2135-2139, 1999).These studies have described that aging is associated with the emergenceof a subset of T helper cells that are characterized by the loss of CD28expression. CD4⁺ CD28⁻ T cells are long lived, typically undergo clonalexpansion in vivo, and react to auto-antigens in vitro. The loss of CD28expression is correlated with a lack of CD40 ligand expression renderingthese CD4⁺ T cells incapable of promoting B cell differentiation andimmunoglobulin secretion. Aging-related accumulation of CD4⁺ CD28⁻ Tcells results in an immune compartment that is skewed towardsauto-reactive responses and away from the generation of high-affinity Bcell responses against exogenous antigens.

BRIEF SUMMARY OF THE INVENTION

One aspect of the present invention provides for a method foreliminating at least a substantial portion of a clonal T cell populationfrom a mixed population of T cells from an individual, comprising,providing a population of cells wherein at least a portion thereofcomprises T cells; exposing the population of cells ex vivo to one ormore pro-apoptotic compositions wherein said exposure induces apoptosisin at least a portion of the T cells; thereby eliminating at least asubstantial portion of said clonal T cells from the mixed population.

The present invention provides a method for eliminating at least asubstantial portion of a clonal T cell subpopulation from a mixedpopulation of T cells from an individual, comprising, exposing apopulation of cells, wherein at least a portion thereof comprises Tcells, to one or more pro-apoptotic or growth inhibiting compositionswherein said exposure induces apoptosis or inhibits growth in at least asubstantial portion of at least one clonal T cell population present inthe mixed population of T cells thereby eliminating at least asubstantial portion of said clonal T cell population from the mixedpopulation of T cells. In one embodiment, the method further comprisesexpanding the mixed population of T cells, by exposing the remainingmixed population of T cells to the pro-apoptotic composition, whereinsaid exposure induces proliferation in the mixed population of T cells.In one particular embodiment, the pro-apoptotic composition comprisesanti-CD3 and anti-CD28 antibodies co-immobilized on a bead. In certainembodiments, the pro-apoptotic composition used to eliminate at least asubstantial portion of said clonal T cell population from the mixedpopulation of T cells is the same composition used to expand theremaining mixed population of T cells.

In one embodiment, the method further comprises expanding the remainingpopulation of cells. In another embodiment, the method further comprisesexpanding the remaining population of cells by exposing the remainingpopulation of cells to a surface wherein the surface has attachedthereto one or more agents that ligate a cell surface moiety of at leasta portion of the remaining T cells and stimulates said remaining Tcells. In a related embodiment, the surface has attached thereto a firstagent that ligates a first T cell surface moiety of a T cell, and thesame or a second surface has attached thereto a second agent thatligates a second moiety of said T cell, wherein said ligation by thefirst and second agent induces proliferation of said T cell.

In one embodiment, the agent attached to the surface is an antibody oran antibody fragment. In another embodiment, the first agent is anantibody or a fragment thereof, and the second agent is an antibody or afragment thereof. In one embodiment the first and the second agents aredifferent antibodies. In one particular embodiment, the first agent isan anti-CD3 antibody; an anti-CD2 antibody, or an antibody fragment ofan anti-CD3 or anti-CD2 antibody. In another embodiment, the secondagent is an anti-CD28 antibody or antibody fragment thereof. In afurther embodiment, the first agent is an anti-CD3 antibody and thesecond agent is an anti-CD28 antibody.

In another embodiment, the cells are exposed to the surfaces of thepresent invention for a time sufficient to increase polyclonality. Incertain embodiments, this increase in polyclonality comprises a shiftfrom mono to oligoclonality or to polyclonality of the T cell populationas measured by a Vβ, Vα, Vγ, or Vδ spectratype profile of at least oneVβ, Vα, Vγ, or Vδ family gene.

Illustrative pro-apoptotic compositions of the present invention includebut are not limited to anti-CD3 antibody, anti-CD2 antibody, anti-CD28antibody, anti-CD20 antibody, target antigen, MHC-peptide tetramers, Fasligand, anti-Fas antibody, IL-2, IL-4, TRAIL, rolipram, doxorubicin,chlorambucil, fludarabine, cyclophosphamide, azathioprine, methotrexate,cyclosporine, mycophenolate, FK506, inhibitors of bcl-2, topoisomeraseinhibitors, interleukin-1β converting enzyme (ICE)-binding agents,Shigella IpaB protein, staurosporine, ultraviolet irradiation, gammairradiation, tumor necrosis factor, target antigens nucleic acidmolecules, proteins or peptides, and non-protein or non-polynucleotidecompounds. In certain embodiments, one or more of these compositions areused at the same time.

In certain embodiments of the present invention, the pro-apoptoticcompositions comprises an autoantigen. Illustrative autoantigens of thepresent invention include but are not limited to, myelin basic protein(MBP), MBP 84-102, MBP 143-168, pancreatic islet cell antigens,collagen, CLIP-170, thyroid antigens, nucleic acid, acetylcholinereceptor, S Antigen, and type II collagen.

The present invention further provides a population of T cells generatedaccording to any of the methods described above.

The present invention provides a method for eliminating at least asubstantial portion of a clonal T cell subpopulation from a mixedpopulation of T cells from an individual, comprising, exposing apopulation of cells, wherein at least a portion thereof comprises Tcells, to one or growth inhibiting compositions wherein said exposureinhibits growth in at least a substantial portion of at least one clonalT cell population present in the mixed population of T cells; the methodfurther comprises expanding the mixed population of T cells, by exposingthe population of cells that is not growth inhibited, i.e., theremaining mixed population of T cells to a surface having attachedthereto one or more agents that bind to a cell surface molecule. In oneembodiment said surface comprises anti-CD3 and anti-CD28 antibodiesco-immobilized on a bead.

One aspect of the present invention provides for methods for treatingautoimmune disease in a patient comprising administering to a patientthe populations of T cells of the present invention. In one embodimentthe patient has been treated with a chemotherapeutic agent prior toadministering the population of T cells. Illustrative chemotherapeuticagents of the present invention include but are not limited to campath,anti-CD3 antibodies, cytoxin, fludarabine, cyclosporine, FK506,mycophenolic acid, steroids, FR901228, and irradiation. In certainembodiments, the patient is treated with a T cell ablative therapy priorto administration of the populations of T cells of the presentinvention.

One aspect of the present invention is a method for eliminating at leasta substantial portion of a clonal T cell population from a population ofT cells from an individual, comprising, providing a population of cellswherein at least a portion thereof comprises T cells; exposing thepopulation of cells to one or more agents that sensitize at least aportion of the T cells to further activation or stimulation, exposingthe population of cells to a surface wherein the surface has attachedthereto one or more agents that ligate a cell surface moiety of at leasta portion of the sensitized T cells and stimulates said sensitized Tcells, wherein the exposure of said sensitized T cells to said surfaceis for a time sufficient to induce apoptosis of said sensitized T cells;thereby eliminating said sensitized T cells from the population. In oneembodiment, the method further comprises exposing said population ofcells to said surface for a time sufficient to stimulate at least aportion of the remaining T cells and wherein said at least a portion ofthe remaining cells proliferates. In a further embodiment, the methodprovides that said surface has attached thereto a first agent thatligates a first T cell surface moiety of a T cell; and the same or asecond surface has attached thereto a second agent that ligates a secondmoiety of said T cell, wherein said ligation by the first and secondagent induces proliferation of said T cell. In one embodiment, at leastone agent is an antibody or an antibody fragment. In another embodiment,the first agent is an antibody or a fragment thereof, and the secondagent is an antibody or a fragment thereof. In yet another embodiment,the first and the second agents are different antibodies. In a relatedembodiment, the first agent is an anti-CD3 antibody, an anti-CD2antibody, or an antibody fragment of an anti-CD3 or anti-CD2 antibody.In yet another embodiment, the second agent is an anti-CD28 antibody orantibody fragment thereof. In another embodiment, the first agent is ananti-CD3 antibody and the second agent is an anti-CD28 antibody.

In a related embodiment, cells are exposed to said surface for a timesufficient to increase polyclonality. In another embodiment, theincrease in polyclonality comprises a shift from mono to oligoclonalityor to polyclonality of the T cell population as measured by a Vβ, Vα,Vγ, or Vδ spectratype profile of at least one Vβ, Vα, Vγ, or Vδ familygene.

In certain embodiments, the patient requires a hematopoietic stem celltransplant. In a related embodiment, the composition that sensitizesrecipient PBMCs that have been treated such that they are unable tocontinue dividing and the population of cells comprises donor T cells.The present invention also provides for populations of T cells generatedaccording to the above methods. The present invention also providesmethods for reducing the risk of, or the severity of, an adverse GVHDeffect in a patient who is undergoing a hematopoietic stem celltransplant, comprising administering to said patient the population of Tcells according to the methods described herein.

In certain embodiments, the patients to receive the cells of the presentinvention require an organ transplant. In a related embodiment thecomposition that sensitizes comprises irradiated donor cells and thepopulation of cells comprises recipient T cells. The present inventionalso provides for a population of cells generated according to thismethod. In one embodiment, these cells are administered to a patientreceiving an organ transplant to reduce the risk of organ rejection. Ina related embodiment, the organ transplant patient is treated with a Tcell ablative therapy prior to administration of the population of Tcells.

In one aspect of the present invention the composition that sensitizescomprises an autoantigen. Illustrative autoantigens of the presentinvention include but are not limited to myelin basic protein (MBP), MBP84-102, MBP 143-168, pancreatic islet cell antigens, S Antigen, and typeII collagen. In one embodiment of the present invention, a patient withan autoimmune disease is treated by administration of a population of Tcells generated according to this method. In a related embodiment, thepatient is treated with a T cell ablative therapy prior to administeringthe population of T cells.

The present invention also provides a method for eliminating a clonal Bcell population from a population of B cells from an individual,comprising, providing a population of cells wherein at least a portionthereof comprises B cells; exposing the population of cells to one ormore pro-apoptotic compositions wherein said exposure induces apoptosisin at least a portion of the B cells; thereby eliminating said portionof B cells from the population. In one embodiment, the method furthercomprises exposing the remaining population of cells to a surfacewherein the surface has attached thereto one or more agents that ligatea cell surface moiety of at least a portion of the remaining B cells andstimulates said remaining B cells. In certain embodiments, thepro-apoptotic composition comprises an autoantigen.

The present invention also provides for compositions of B-cellsgenerated according to the above methods.

In one embodiment of the present invention, a patient with an autoimmunedisease is treated with a composition comprising the populations ofB-cells generated using the methods of the present invention. In arelated embodiment, the patient is treated with a B cell ablativetherapy prior to administering the population of B cells.

One aspect of the present invention provides methods for generating asubstantially pure population of T cells from a population of T cellsfrom an individual, comprising providing a population of cells whereinat least a portion thereof comprises T cells: exposing the population ofT cells ex vivo to a composition that preferentially selects and/orstimulates surface CD3⁺ and CD28⁺ molecules, thereby generating asubstantially pure population of CD3⁺/CD28⁺ T cells. In a relatedembodiment the population of pure T cells generated is a substantiallypure population of CD4⁺/CD3⁺/CD28⁺ T cells. In a related embodiment thepopulation of pure T cells is a substantially pure population ofCD8⁺/CD3⁺/CD28⁺ T cells.

In one aspect of the invention the purity of the CD3⁺/CD28⁺ T cells isat least 90% pure. In further embodiments, the purity of the CD3⁺/CD28⁺T cells is 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% pure. In anotherembodiment the purity of the CD3⁺/CD28⁺ T cells is at least 99% pure. Ina related embodiment the purity of the CD3⁺/CD28⁺ T cells is at least99.9% pure. Therefore, one aspect of the present invention is apopulation of CD3⁺/CD28⁺ T cells comprising less than 10% of CD28−cells. In certain embodiments, the population of CD3⁺/CD28⁺ T cellscomprises less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1%contaminating CD28⁻ T cells.

In one embodiment the CD3⁺ surface molecule is stimulated using anti-CD3antibodies and the CD28⁺ surface molecule is stimulated using anti-CD28antibodies.

Therefore, the present invention also provides methods for thegeneration of a substantially pure population of CD3⁺ CD28⁺ T cells,including CD4⁺ CD3⁺CD28⁺ T cells, and CD8⁺ CD3⁺CD28⁺ T cells. These Tcell populations could then be used in the treatment of people sufferingfrom autoimmune diseases such as, rheumatoid arthritis, multiplesclerosis, insulin dependent diabetes, Addison's disease, celiacdisease, chronic fatigue syndrome, inflammatory bowel disease,ulcerativecolitis, Crohn's disease, Fibromyalgia, systemic lupuserythematosus, psoriasis, Sjogren's syndrome, hyperthyroidism/Gravesdisease, hypothyroidism/Hashimoto's disease, Insulin-dependent diabetes(type 1), Myasthenia Gravis, endometriosis, scleroderma, perniciousanemia, Goodpasture syndrome, Wegener's disease, glomerulonephritis,aplastic anemia, paroxysmal nocturnal hemoglobinuria, myelodysplasticsyndrome, idiopathic thrombocytopenic purpura, autoimmune hemolyticanemia, Evan's syndrome, Factor VIII inhibitor syndrome, systemicvasculitis, dermatomyositis, polymyositis, pemphigus vulgaris (PV),paraneoplastic pemphigus (PNP), and rheumatic fever.

The present invention further provides a method for activating andexpanding a population of T cells by cell surface moiety ligation,comprising providing a population of cells wherein at least a portionthereof comprises T cells, contacting the population of cells with asurface, wherein the surface has attached thereto one or more agentsthat ligate a cell surface moiety of at least a portion of the T cellsand stimulates said T cells, wherein said surface is present at a ratioof said surface to said cells such that at least a substantial portionof at least one population of antigen-specific T cells is deleted afterabout 8 days of culture. In one embodiment of the invention, the ratiois from about 50:1 to about 5:1. In certain embodiments, the ratio isfrom about 100:1 to about 2:1. In one embodiment the ratio is at leastabout 45:1. In certain embodiments, the ratio is at least about 40:1,35:1, 30:1, 25:1, 20:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1,7:1, 6:1, 5:1, 4:1, 3:1, or 2:1. In one particular embodiment the ratiois about 5:1.

The present invention provides a method for eliminating at least asubstantial portion of a clonal T cell subpopulation from a mixedpopulation of T cells from an individual, comprising, exposing apopulation of cells, wherein at least a portion thereof comprises Tcells, to one or more pro-apoptotic compositions wherein said exposureinduces apoptosis in at least a substantial portion of at least oneclonal T cell population present in the mixed population of T cellsthereby eliminating at least a substantial portion of said clonal T cellpopulation from the mixed population of T cells.

The present invention further provides methods for improved transplantefficacy by administration of XCELLERATED™ T cells following high-dosechemotherapy and autologous stem cell transplantation.

The present invention also provides a method for treating a patientafflicted with an autoimmune disease comprising contacting a populationof cells from the patient, wherein at least a portion thereof comprisesT cells, with a surface, wherein said surface has attached thereto oneor more agents that ligate a cell surface moiety of at least a portionof the T cells and stimulates said T cells, wherein said surface ispresent at a ratio of said surface to said cells such that at least asubstantial portion of at least one population of antigen-specific Tcells is deleted after about 8 days of culture; and administering to thepatient an effective amount of T cells from (a) such that in vivohomeostatic proliferation is inhibited; thereby treating autoimmunedisease. In certain embodiments, the ratio is from about 10:1 to about5:1.

The present invention further provides a method for treating a patientafflicted with an autoimmune disease comprising contacting a populationof cells from the patient, wherein at least a portion thereof comprisesT cells, with a surface, wherein said surface has attached thereto oneor more agents that ligate a cell surface moiety of at least a portionof the T cells and stimulates said T cells; administering to the patientthe T cells of (a) at a dose such that homeostatic proliferation ofendogenous T cells is inhibited; thereby treating autoimmune disease.

The present invention also provides a method for treating a patientinfected with HIV comprising contacting a population of cells from thepatient, wherein at least a portion thereof comprises T cells, with asurface, wherein said surface has attached thereto one or more agentsthat ligate a cell surface moiety of at least a portion of the T cellsand stimulates said T cells, wherein said surface is present at a ratioof said surface to said cells such that at least a substantial portionof HIV-infected T cells is deleted after about 8 days of culture; andadministering to the patient an effective amount of T cells from (a)such that in vivo homeostatic proliferation is inhibited; therebytreating the patient infected with HIV.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 is a dot plot showing the presence of CD3+ CD8+HLA-A2CMVpp65antigen specific T cells in an HLA-A2-positive donor.

FIG. 2 is a dot plot showing an increase in CD25 expression inCMV-activated HLA-A2CMVpp65 antigen-specific T cells.

FIG. 3 is a dot plot showing the upregulation of CD25 on restimulatedcells, and the deletion of prestimulated tetramer-positive cells (i.e.,CMVpp65-Ag-specific) by the secondary strong stimulation provided by the3×28 beads. At day 14 post-primary stimulation, cultures were eitherleft unstimulated (Panels A1-A4) or were restimulated using theXCELLERATE™ process with 3×28 beads for 16 hours (Panels B1-B4). CD25 isupregulated on restimulated cells (Panel B2), but tetramer-positive(i.e., CMVpp65-Ag-specific) prestimulated cells were deleted by thesecondary strong stimulation provided by the 3×28 beads (Panel B3).

FIG. 4 is a histogram showing the increase in expression of key effectormolecules, including CD95, on leukemic B-cells co-cultured withXCELLERATED T cells™.

FIG. 5 is a dot plot showing the induction of apoptosis in leukemicB-cells co-cultured with XCELLERATED T cells™.

FIG. 6 is a graph showing the disappearance of leukemic B-cells duringthe XCELLERATE™ process and the concomitant expansion of T cells.

FIG. 7 is a graph comparing fold increase of polyclonal T cells to thefold increase of CMV pp65 A2-tetramer+ (antigen-specific) T cells usingvarying bead:cell ratios. Solid bars represent polyclonal T cells.Striped bars represent CMV-specific T cells.

FIG. 8 shows the spectratype analysis of T cells from a rheumatoidarthritis patient pre-XCELLERATE™(left panels) and post-XCELLERATE™(right panels) (5:1 bead:T cell ratio). Restoration of healthy T cellrepertoire was observed (see in particular TCRBV 13a and TCRBV 3 panels,pre and post XCELLERATE™).

FIG. 9 is a bar graph showing the Th1 phenotype of XCELLERATED™ T cellsfrom patients with scleroderma, Crohn's Disease, lupus, and rheumatoidarthritis.

FIG. 10 is a 4 panel dot plot showing the deletion of islet-specificCD8+ autoreactive T cells in a mouse diabetes model. Islet-specific Tcells were detected using flow cytometry and MHC-class I tetramerstaining.

DETAILED DESCRIPTION OF THE INVENTION

Prior to setting forth the invention, it may be helpful to anunderstanding thereof to set forth definitions of certain terms thatwill be used hereinafter.

The term “biocompatible”, as used herein, refers to the property ofbeing predominantly non-toxic to living cells.

The term “stimulation”, as used herein, refers to a primary responseinduced by ligation of a cell surface moiety. For example, in thecontext of receptors, such stimulation entails the ligation of areceptor and a subsequent signal transduction event. With respect tostimulation of a T cell, such stimulation refers to the ligation of a Tcell surface moiety that in one embodiment subsequently induces a signaltransduction event, such as binding the TCR/CD3 complex. Further, thestimulation event may activate a cell and up- or down-regulateexpression of cell surface molecules such as receptors or adhesionmolecules, or up- or down-regulate secretion of a molecule, such asdownregulation of Tumor Growth Factor beta (TGF-β). Thus, ligation ofcell surface moieties, even in the absence of a direct signaltransduction event, may result in the reorganization of cytoskeletalstructures, or in the coalescing of cell surface moieties, each of whichcould serve to enhance, modify, or alter subsequent cell responses.

The term “activation”, as used herein, refers to the state of a cellfollowing sufficient cell surface moiety ligation to induce a measurablemorphological, phenotypic, and/or functional change. Within the contextof T cells, such activation may be the state of a T cell that has beensufficiently stimulated to induce cellular proliferation. Activation ofa T cell may also induce cytokine production and/or secretion, and up-or down-regulation of expression of cell surface molecules such asreceptors or adhesion molecules, or up- or down-regulation of secretionof certain molecules, and performance of regulatory or cytolyticeffector functions. Within the context of other cells, this term inferseither up- or down-regulation of a particular physico-chemical process.

The term “target cell”, as used herein, refers to any cell that isintended to be stimulated by cell surface moiety ligation.

An “antibody”, as used herein, includes both polyclonal and monoclonalantibodies (mAb); primatized (e.g., humanized); murine; mouse-human;mouse-primate; and chimeric; and may be an intact molecule, a fragmentthereof (such as scFv, Fv, Fd, Fab, Fab′ and F(ab)′₂ fragments), ormultimers or aggregates of intact molecules and/or fragments; and mayoccur in nature or be produced, e.g., by immunization, synthesis orgenetic engineering; an “antibody fragment,” as used herein, refers tofragments, derived from or related to an antibody, which bind antigenand which in some embodiments may be derivatized to exhibit structuralfeatures that facilitate clearance and uptake, e.g., by theincorporation of galactose residues. This includes, e.g., F(ab),F(ab)′₂, scFv, light chain variable region (V_(L)), heavy chain variableregion (V_(H)), and combinations thereof.

The term “protein”, as used herein, includes proteins, glycoproteins andother cell-derived modified proteins, polypeptides and peptides; and maybe an intact molecule, a fragment thereof, or multimers or aggregates ofintact molecules and/or fragments; and may occur in nature or beproduced, e.g., by synthesis (including chemical and/or enzymatic) orgenetic engineering.

The term “agent”, “ligand”, or “agent that binds a cell surface moiety”,as used herein, refers to a molecule that binds to a defined populationof cells. The agent may bind any cell surface moiety, such as areceptor, an antigenic determinant, or other binding site present on thetarget cell population. The agent may be a protein, peptide, antibodyand antibody fragments thereof, fusion proteins, synthetic molecule, anorganic molecule (e.g., a small molecule), or the like. Within thespecification and in the context of T cell stimulation, antibodies areused as a prototypical example of such an agent.

The term “cell surface moiety” as used herein may refer to a cellsurface receptor, an antigenic determinant, or any other binding sitepresent on a target cell population.

The terms “agent that binds a cell surface moiety” and “cell surfacemoiety”, as used herein, should be viewed as acomplementary/anti-complementary set of molecules that demonstratespecific binding, generally of relatively high affinity.

A “co-stimulatory signal”, as used herein, refers to a signal, which incombination with a primary signal, such as TCR/CD3 ligation, leads to Tcell proliferation and/or activation.

“Separation”, as used herein, includes any means of substantiallypurifying one component from another (e.g., by filtration, affinity,buoyant density, or magnetic attraction).

A “surface”, as used herein, refers to any surface capable of having anagent attached thereto and includes, without limitation, metals, glass,plastics, co-polymers, colloids, lipids, cell surfaces, and the like.Essentially any surface that is capable of retaining an agent bound orattached thereto.

“Monoclonality”, as used herein, in the context of a population of Tcells, refers to a population of T cells that has a single specificityas defined by spectratype analysis (a measure of the TCR Vβ, Vα, Vγ, orVδ chain hypervariable region repertoire). A population of T cells isconsidered monoclonal (or mono-specific) when the Vβ, Vα, Vγ, and/or Vδspectratype profile for a given TCR Vβ, Vα, Vγ, and/or Vδ family has asingle predominant peak. Spectratype analysis distinguishes rearrangedvariable genes of a particular size, not sequence. Thus, it isunderstood that a single peak could represent a population of T cellsexpressing any one of a limited number of rearranged TCR variable genes(Vβ, Vα, Vγ, or Vδ) comprising any one of the 4 potential nucleotides(adenine (a), guanine (g), cytosine (c), or thymine (t)) or acombination of the 4 nucleotides at the junctional region. In certainembodiments of the present invention, it may be desirable to clone andsequence a particular band to determine the sequence(s) of therearranged variable gene(s) present in the band representing aparticular length.

“Oligoclonality”, as used herein, in the context of a population of Tcells, refers to a population of T cells that has multiple, but narrowantigen specificity. This can be defined by spectratype analysis (ameasure of the TCR Vβ, Vα, Vγ, or Vδ) chain hypervariable regionrepertoire). A population of T cells is considered oligoclonal when theVβ spectratype profile for a given TCR Vβ, Vα, Vγ, or Vδ family hasbetween about 2 and about 4 predominant peaks. This can also be definedby generation and characterization of antigen-specific clones to anantigen of interest.

“Polyclonality”, as used herein, in the context of a population of Tcells, refers to a population of T cells that has multiple and broadantigen specificity. This can be by spectratype analysis (a measure ofthe TCR Vβ, Vα, Vγ, or Vδ chain hypervariable region repertoire). Apopulation of T cells is considered polyclonal when the Vβspectratypeprofile for a given TCR Vβ, Vα, Vγ, or Vδ family has multiple peaks,typically 5 or more predominant peaks and in most cases with Gaussiandistribution. Polyclonality can also be defined by generation andcharacterization of antigen-specific clones to an antigen of interest.

“Restoring or increasing the polyclonality”, as used herein refers to ashift from a monoclonal profile to an oligoclonal profile or to apolyclonal profile, or from an oligoclonal profile to a polyclonalprofile, in expressed TCR Vβ, Vα, Vγ, and/or Vδ genes in a population ofT cells, as measured by spectratype analysis or by similar analysis suchas flow cytometry or sequence analysis. The shift from a monoclonal Vβ,Vα, Vγ, and/or Vδ expression profile in a population of T cells to anoligoclonal profile or to a polyclonal profile is generally seen in atleast one TCR Vβ, Vα, Vγ, and/or Vδ family. In one embodiment of thepresent invention, this shift is observed in 2, 3, 4, or 5 Vβfamilies.In certain embodiments of the present invention, a shift is observed in6, 7, 8, 9, or 10 Vβfamilies. In a further embodiment of the presentinvention, a shift is observed in from 11, 12, 13, or 14 Vβ families. Ina further embodiment of the present invention, a shift is observed infrom 15 to 20 Vβ families. In a further embodiment of the presentinvention, a shift is observed in 20 to 24 Vβ families. In anotherembodiment, a shift is seen in all Vβ families. The functionalsignificance of restoring or increasing the polyclonality of apopulation of T cells is that the immune potential, or the ability torespond to a full breadth of antigens, of the population of T cells isrestored or increased. In certain aspects of the present invention, someT cells within a population may not have their TCRs engaged by themethods set forth herein (e.g., T cells with downregulated TCRexpression). However, by being in close proximity to T cells activatedby the methods described herein, and the factors secreted by them, theseT cells may in turn upregulate their TCR expression thereby resulting ina further increase in the polyclonality of the population of T cells.Restoration or increase in polyclonality can also be measured bydetermining the breadth of response to a particular antigen of interest,for example by measuring the number of different epitopes recognized byantigen-specific cells. This can be carried out using standardtechniques for generating and cloning antigen-specific T cells in vitro.

The term “clonal T cell population” as used herein, refers to a T cellpopulation that has a given range of specificities against a giventarget antigen. This can be measured by any number of assays known inthe art, for example by generating and measuring the breadth ofspecificities (i.e. number of different specificities) ofantigen-specific clones in a given population. A clonal T cellpopulation can also be defined by having either monoclonal oroligoclonal specificity as defined by spectratype analysis (a measure ofthe TCR Vβ, Vα, Vγ, or Vδ chain hypervariable region repertoire).

The term “animal” or “mammal” as used herein, encompasses all mammals,including humans. Preferably, the animal of the present invention is ahuman subject.

The term “exposing” as used herein, refers to bringing into the state orcondition of immediate proximity or direct contact.

The term “proliferation” as used herein, means to grow or multiply byproducing new cells.

“Immune response or responsiveness” as used herein, refers to activationof cells of the immune system, including but not limited to, T cells,such that a particular effector function(s) of a particular cell isinduced. Effector functions may include, but are not limited to,proliferation, secretion of cytokines, secretion of antibodies,expression of regulatory and/or adhesion molecules, and the ability toinduce cytolysis.

“Stimulating an immune response” as used herein, refers to anystimulation such that activation and induction of effector functions ofcells of the immune system are achieved.

“Immune response dysfunction” as used herein, refers to theinappropriate activation and/or proliferation, or lack thereof, of cellsof the immune system, and/or the inappropriate secretion, or lackthereof, of cytokines, and/or the inappropriate or inadequate inductionof other effector functions of cells of the immune system, such asexpression of regulatory, adhesion, and/or homing receptors, and theinduction of cytolysis.

“Particles” or “surface” as used herein, may include a colloidalparticle, a microsphere, nanoparticle, a bead, or the like. A surfacemay be any surface capable of having a ligand bound thereto orintegrated into, including cell surfaces (for example K562 cells), andthat is biocompatible, that is, substantially non-toxic to the targetcells to be stimulated. In the various embodiments, commerciallyavailable surfaces, such as beads or other particles, are useful (e.g.,Miltenyi Particles, Miltenyi Biotec, Germany; Sepharose beads, PharmaciaFine Chemicals, Sweden; DYNABEADS™, Dynal Inc., New York; PURABEADS™,Prometic Biosciences, magnetic beads from Immunicon, Huntingdon Valley,Pa., microspheres from Bangs Laboratories, Inc., Fishers, Ind.).

“Paramagnetic particles” as used herein, refer to particles, as definedabove, that localize in response to a magnetic field.

A “pro-apoptotic composition” “apoptotic compositions” or “inducer ofapoptosis”, as used herein refers to any composition or stimulus thatincreases the apoptotic activity of a cell either when administeredalone or in conjunction with other pro-apoptotic compositions. Thepro-apoptotic compositions used in the methods of the present inventionpreferably induce apoptosis in activated T cells, NKT, NK or B-cells. Incertain embodiments, a pro-apoptotic composition of the presentinvention will induce apoptosis without further activation/stimulation.Illustrative examples of such compositions or stimuli include, but arenot limited to, deprivation of a growth factor, oxidizing conditions,heat stress, serum starvation, phorbol myristate acetate (PMA) andionomycin, superantigens (e.g. SEA, SEB, and the like) variousantibodies, such as anti-CD2, anti-CD3, anti-CD28, anti-CD20, anti-Fasantibody, or any combination thereof, MHC-peptide tetramers or dimers,Fas ligand, IL-2, IL-4, TRAIL, rolipram, doxorubicin, chlorambucil,fludarabine, corticosteroids, glucocorticoids, cyclosporine,cyclophosphamide, FK506, azathioprine, methotrexate, mycophenolate,annexin, caspases, inhibitors of bcl-2, topoisomerase inhibitors,interleukin-1β converting enzyme (ICE)-binding agents, Shigella IpaBprotein, staurosporine, ultraviolet irradiation, gamma irradiation,radiation, tumor necrosis factor, various histone deacetylaseinhibitors, and others well known in the art. In certain embodiments,the pro-apoptotic compositions comprises a surface, such as a magneticbead, having attached thereto one or more agents that binds a cellsurface moiety. In this regard, the agent can be any agent as describedherein. In one embodiment, the surface has attached thereto at leastanti-CD3 antibodies. In another embodiment, the surface has attachedthereto anti-CD3 and anti-CD28 antibodies. In addition, a stimulator ofapoptosis can be a polypeptide that is capable of increasing or inducingthe apoptotic activity of a cell. Such polypeptides include those thatdirectly regulate the apoptotic pathway such as Bax, Bad, Bcl-xS, Bak,Bik, and active caspases as well as those that indirectly regulate thepathway. In certain embodiments, the pro-apoptotic composition comprisesactivated T cells, such as XCELLERATED T cells™ (such as those describedin U.S. patent application Ser. No. 10/133,236), in particular forinducing apoptosis in populations of B-cells. Other illustrativepro-apoptotic compositions include, but are not limited to, irradiatedcells (e.g. donor or recipient (allogeneic) cells), target antigens(e.g. defined autoimmune target antigens for example, in multiplesclerosis, the target antigen identified as myelin basic protein (MBP)MBP 84-102, or MBP 143-168; pancreatic islet cell antigens; in uveitis,the S Antigen; or in rheumatoid arthritis, type II or other types ofcollagen; in Grave's disease, thyroid receptor; in Myasthena gravis,acetylcholine receptor), cytoplasmic linker protein-170 (CLIP-170),nucleic acid molecules, proteins or peptides, and non-protein ornon-polynucleotide compounds.

A “composition that sensitizes cells to further activation orstimulation” or “sensitizing composition” as used herein is anycomposition which sensitizes cells to subsequent activation/stimulation.Upon subsequent activation/stimulation, sensitized cells undergoapoptosis. Sensitizing compositions of the present invention alsosensitize cells to the effects of pro-apoptotic compositions.Illustrative compositions that sensitize cells to further activation,stimulation, or the effects of pro-apoptotic compositions include cellsthat have been treated such that they are unable to continue dividing,for example by irradiation, (e.g. donor or recipient (allogeneic)cells), superantigens (e.g. SEA, SEB, and the like), target antigens(e.g. defined autoimmune target antigens for example, in multiplesclerosis, the target antigen identified as myelin basic protein (MBP)MBP 84-102, or MBP 143-168; pancreatic islet cell antigens; in uveitis,the S Antigen; or in rheumatoid arthritis, type II or other types ofcollagen; in Grave's disease, thyroid receptor; in Myasthena gravis,acetylcholine receptor, nucleic acid molecules, proteins or peptides,and non-protein or non-polynucleotide compounds), protein, glycoprotein,peptides, antibody/antigen complexes, cell lysate, non-soluble celldebris, apoptotic bodies, necrotic cells, whole cells from a cell linethat have been treated such that they are unable to continue dividing,natural or synthetic complex carbohydrates, lipoproteins, transformedcells or cell line, transfected cells or cell line, or transduced cellsor cell line, or any combination thereof.

Apoptosis, for purposes of the present invention, is defined asprogrammed cell death. Apoptosis is a programmed cell death which is awidespread phenomenon that plays a crucial role in the myriad ofphysiological and pathological processes. Apoptosis occurs inembryogenesis, metamorphosis, endocrine-dependent tissue atrophy, normaltissue turnover, and death of immune thymocytes (induced through theirantigen-receptor complex or by glucocorticoids) (Itoh et al., Cell66:233, 1991). During maturation of T cells in the thymus, T cells thatrecognize self-antigens are destroyed through the apoptotic process,whereas others are positively selected. The possibility that some Tcells recognizing certain self epitopes (e.g., inefficiently processedand presented antigenic determinants of a given self protein) escapethis elimination process and subsequently play a role in autoimmunediseases has been suggested (Gammon et al., Immunology Today 12:193,1991). Necrosis is an accidental cell death which is the cell's responseto a variety of harmful conditions and toxic substances. Apoptosis,morphologically distinct from necrosis, is a spontaneous form of celldeath that occurs in many different tissues under various conditions.Apoptosis occurs in two stages. The cell undergoes nuclear andcytoplasmic condensation, and may eventually break into a number ofmembrane-bound fragments containing structurally intact apoptoticbodies, which are phagocytosed by neighboring cells and rapidlydegraded. Alternatively, cells entering the apoptotic pathway may bephagocytosed prior to degeneration into membrane bound bodies. Apoptosisis observed in many different tissues, healthy and neoplastic, adult andembryonic. Death occurs spontaneously, or is induced by physiological ornoxious agents. Apoptosis is a basic physiological process that plays amajor role in the regulation of cell populations.

Methods for measuring apoptosis are well known in the art. Apoptosis canbe determined by methods such as, for example, DNA ladder, electron orlight microscopy, flow cytometry, and different commercially availablekits for the determination of apoptosis.

As used herein, a “growth inhibiting composition” is any substance thatinhibits growth in cells, or otherwise renders cells dysfunctional andunable to divide either when administered alone or in conjunction withother compositions of the present invention. The growth-inhibitingcompositions used in the methods of the present invention preferablyinhibit growth in activated T cells, NKT, NK or B-cells. Illustrativeexamples of such compositions or stimuli include, but are not limitedto, but are not limited to, deprivation of a growth factor, oxidizingconditions, heat stress, serum starvation, phorbol myristate acetate(PMA) and ionomycin, superantigens (e.g. SEA, SEB, and the like) variousantibodies, such as anti-CD2, anti-CD3, anti-CD28, anti-CD20, anti-Fasantibody, or any combination thereof, MHC-peptide tetramers or dimers,Fas ligand, IL-2, IL-4, TRAIL, rolipram, doxorubicin, chlorambucil,fludarabine, corticosteroids, glucocorticoids, cyclosporine,cyclophosphamide, FK506, azathioprine, methotrexate, mycophenolate,annex in, caspases, inhibitors of bcl-2, topoisomerase inhibitors,interleukin-1β converting enzyme (ICE)-binding agents, Shigella IpaBprotein, staurosporine, ultraviolet irradiation, gamma irradiation,radiation, tumor necrosis factor, various histone deacetylaseinhibitors, and others well known in the art. In certain embodiments,the growth inhibiting compositions comprises a surface, such as amagnetic bead, having attached thereto one or more agents that binds acell surface moiety. In this regard, the agent can be any agent asdescribed herein. In one embodiment, the surface has attached thereto atleast anti-CD3 antibodies. In another embodiment, the surface hasattached thereto anti-CD3 and anti-CD28 antibodies. In addition, agrowth inhibiting composition can comprise a polypeptide that is capableof inhibiting growth of a cell. Such polypeptides include those peptidessuch as Bax, Bad, Bcl-xS, Bak, Bik, and active caspases. Otherillustrative growth inhibiting compositions include, but are not limitedto, irradiated cells (e.g. donor or recipient (allogeneic) cells),target antigens (e.g. defined autoimmune target antigens for example, inmultiple sclerosis, the target antigen identified as myelin basicprotein (MBP) MBP 84-102, or MBP 143-168; pancreatic islet cellantigens; in uveitis, the S Antigen; or in rheumatoid arthritis, type IIor other types of collagen; in Grave's disease, thyroid receptor; inMyasthena gravis, acetylcholine receptor), cytoplasmic linkerprotein-170 (CLIP-170), nucleic acid molecules, proteins or peptides,and non-protein or non-polynucleotide compounds.

As used herein, a “substantially pure” population of CD3⁺/CD28⁺ T cellsis a population of cells that is comprised of at least about 90%CD3⁺/CD28⁺ T cells. In certain aspects of the invention a “substantiallypure” population of CD3+/CD28+ T cells is a population of cells that iscomprised of at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98%CD3⁺/CD28⁺ T cells, preferably at least about 99%, and even morepreferably about 99.9% or more.

Sources of Mixed Population of Cells

In one embodiment, cells to be exposed to the pro-apoptotic or growthinhibiting compositions and/or sensitizing compositions are from thecirculating blood of an individual and are obtained from one or moreunits of blood or from an apheresis or leukapheresis. The apheresisproduct typically contains lymphocytes, including T cells, monocytes,granulocytes, B cells, other nucleated white blood cells, red bloodcells, and platelets. Prior to exposure to a sensitizing composition andsubsequent activation and/or stimulation, a source of T cells isobtained from a subject. The term “subject” is intended to includeliving organisms in which an immune response can be elicited (e.g.,mammals). Examples of subjects include humans, dogs, cats, mice, rats,and transgenic species thereof. T cells can be obtained from a number ofsources, including peripheral blood mononuclear cells, bone marrow,thymus, tissue biopsy, tumor, lymph node tissue, gut associated lymphoidtissue, mucosa associated lymphoid tissue, spleen tissue, or any otherlymphoid tissue, and tumors. T cells can be obtained from T cell linesand from autologous or allogeneic sources. T cells may also be obtainedfrom a xenogeneic source, for example, from mouse, rat, non-humanprimate, and pig. In certain embodiments of the present invention, Tcells can be obtained from a unit of blood collected from a subjectusing any number of techniques known to the skilled artisan, such asficoll separation. In one preferred embodiment, cells from thecirculating blood of an individual are obtained by apheresis orleukapheresis. The apheresis product typically contains lymphocytes,including T cells, monocytes, granulocytes, B cells, other nucleatedwhite blood cells, red blood cells, and platelets. In one embodiment,the cells collected by apheresis may be washed to remove the plasmafraction and to place the cells in an appropriate buffer or media forsubsequent processing steps. In one embodiment of the invention, thecells are washed with phosphate buffered saline (PBS). In an alternativeembodiment, the wash solution lacks calcium and may lack magnesium ormay lack many if not all divalent cations. As those of ordinary skill inthe art would readily appreciate a washing step may be accomplished bymethods known to those in the art, such as by using a semi-automated“flow-through” centrifuge (for example, the Cobe 2991 cell processor,Baxter) according to the manufacturer's instructions. After washing, thecells may be resuspended in a variety of biocompatible buffers, such as,for example, calcium (Ca)-free, magnesium (Mg)-free PBS. Alternatively,the undesirable components of the apheresis sample may be removed andthe cells directly resuspended in culture media.

In another embodiment, T cells are isolated from peripheral bloodlymphocytes by lysing or removing the red blood cells and depleting themonocytes, for example, by centrifugation through a PERCOLL™ gradient. Aspecific subpopulation of T cells, such as CD28⁺, CD4⁺, CD8⁺, CD45RA⁺,and CD45RO⁺T cells, can be further isolated by positive or negativeselection techniques. For example, in one preferred embodiment, T cellsare isolated by incubation with anti-CD3/anti-CD28 (i.e.,3×28)-conjugated beads, such as DYNABEADS® M-450 CD3/CD28 T, for a timeperiod sufficient for positive selection of the desired T cells. In oneembodiment, the time period is about 30 minutes. In a furtherembodiment, the time period ranges from 30 minutes to 36 hours or longerand all integer values there between. In a further embodiment, the timeperiod is at least 1, 2, 3, 4, 5, or 6 hours. In yet another preferredembodiment, the time period is 10 to 24 hours. In one preferredembodiment, the incubation time period is 24 hours. For isolation of Tcells from patients with leukemia, use of longer incubation times, suchas 24 hours, can increase cell yield. Longer incubation times may beused to isolate T cells in any situation where there are few T cells ascompared to other cell types, such in isolating tumor infiltratinglymphocytes (TIL) from tumor tissue or from immunocompromisedindividuals. Further, use of longer incubation times can increase theefficiency of capture of CD8+ T cells. For example, CD3⁺, CD28⁺ T cellscan be positively selected using CD3/CD28 conjugated magnetic beads(e.g., DYNABEADS® M-450 CD3/CD28 T cell Expander). In one aspect of thepresent invention, enrichment of a T cell population by negativeselection can be accomplished with a combination of antibodies directedto surface markers unique to the negatively selected cells. A preferredmethod is cell sorting and/or selection via negative magneticimmunoadherence or flow cytometry that uses a cocktail of monoclonalantibodies directed to cell surface markers present on the cellsnegatively selected. For example, to enrich for CD4⁺ cells by negativeselection, a monoclonal antibody cocktail typically includes antibodiesto CD14, CD20, CD11b, CD16, HLA-DR, and CD8.

An additional aspect of the present invention provides a T cellpopulation or composition that has been depleted or enriched forpopulations of cells expressing a variety of markers, such as CD62L,CD45RA or CD45RO, cytokines (e.g. IL-2, IFN-γ, IL-4, IL-10), cytokinereceptors (e.g. CD25), perforin, adhesion molecules (e.g. VLA-1, VLA-2,VLA-4, LPAM-1, LFA-1), and/or homing molecules (e.g. L-Selectin), priorto sensitization, stimulation and expansion. In one embodiment, cellsexpressing any of these markers are depleted or positively selected byantibodies or other ligands/binding agents directed to the marker. Oneof ordinary skill in the art would readily be able to identify a varietyof particular methodologies for depleting or positively selecting for asample of cells expressing a desired marker.

Monocyte populations (i.e., CD14⁺ cells) may be depleted from bloodpreparations prior to ex vivo expansion by a variety of methodologies,including anti-CD14 coated beads or columns, or utilization of thephagocytotic activity of these cells to facilitate removal or throughadherence to plastic. Accordingly, in one embodiment, the invention usesparamagnetic particles of a size sufficient to be engulfed byphagocytotic monocytes. In certain embodiments, the paramagneticparticles are commercially available beads, for example, those producedby Dynal AS under the trade name DYNABEADS™. Exemplary DYNABEADS™ inthis regard are M-280, M-450, and M-500. In one aspect, othernon-specific cells are removed by coating the paramagnetic particleswith “irrelevant” proteins (e.g., serum proteins or antibodies).Irrelevant proteins and antibodies include those proteins and antibodiesor fragments thereof that do not specifically target the T cells to beexpanded. In certain embodiments the irrelevant beads include beadscoated with sheep anti-mouse antibodies, goat anti-mouse antibodies, andhuman serum albumin.

In brief such depletion of monocytes is performed by preincubating PBMCthat have been isolated from whole blood using Ficoll, or apheresedperipheral blood with one or more varieties of irrelevant ornon-antibody coupled paramagnetic particles at any amount that allowsfor removal of monocytes (approximately a 20:1 bead:cell ratio)for about30 minutes to 2 hours at 22 to 37 degrees C., followed by magneticremoval of cells which have attached to or engulfed the paramagneticparticles. Preincubation can also be done at temperatures as low as 3-4degrees C. Such separation can be performed using standard methodsavailable in the art. For example, any magnetic separation methodologymay be used including a variety of which are commercially available,(e.g., DYNAL® Magnetic Particle Concentrator (DYNAL MPC®)). Assurance ofrequisite depletion can be monitored by a variety of methodologies knownto those of ordinary skill in the art, including flow cytometricanalysis of CD14 positive cells, before and after said depletion.

T cells for exposure to pro-apoptotic and/or sensitizing compositionsand subsequent stimulation may also be frozen after the washing step,which does not require the monocyte-removal step. Wishing not to bebound by theory, the freeze and subsequent thaw step provides a moreuniform product by removing granulocytes and to some extent monocytes inthe cell population. After the washing step that removes plasma andplatelets, the cells may be suspended in a freezing solution. While manyfreezing solutions and parameters are known in the art and will beuseful in this context, one method involves using PBS containing a finalconcentration of 10% DMSO and 4% human serum albumin, or other suitablecell freezing media, the cells then are frozen to −80° C. at a rate of1° per minute and stored in the vapor phase of a liquid nitrogen storagetank.

Elimination of Undesired Subpopulations of Cells from a Mixed Populationof Cells

Direct Exposure to Pro-Apoptotic Compositions

The present invention provides for methods to eliminate at least aportion of undesired clonal populations of cells, typically T cells, Bcells, NKT, or NK cells, from a population of immune cells. The presentinvention further provides for compositions comprising populations ofcells that no longer contain undesired cells, or have a significantlyreduced number of undesired cells, and uses thereof.

Undesired populations of cells can be eliminated or reduced by astatistically significant amount directly through the exposure of saidcells to a pro-apoptotic composition. Exposure to the pro-apoptoticcomposition can take place in vivo or in vitro. Without being bound bytheory, the previously activated cells are thought to be more sensitiveto apoptotic compositions than naïve or unactivated cells. Therefore,exposure to apoptotic compositions either in vivo or in vitro, usingdoses and conditions that induce apoptosis, will selectively kill highlyactivated cells such as unwanted autoreactive cells in a patient. Inpreferred embodiments of the present invention, the autoreactive cellsto be eliminated comprise T cells, NKT, NK, or B cells.

Thus, the present invention provides methods for the elimination of atleast a substantial portion of any unwanted subpopulation of clonalcells (such as T, B, NKT, or NK cells) from a mixed population of immunecells. For the purposes of the present invention, a substantial portionmeans at least 70% of the unwanted subpopulation of cells. In certainembodiments, a substantial portion means 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, 99% and higher of the unwanted subpopulation of cells.Elimination of cells can be measured using any number of techniquesknown in the art, including but not limited to flow cytometric analysisusing a variety of antibodies and/or peptide-MHC tetramers andfunctional assays such as proliferation and chromium release assays.

Pro-apoptotic compositions or inducers of apoptosis refers to anycomposition or stimulus that increases the apoptotic activity of a celleither when administered alone or in conjunction with (in combinationwith, before or after) other pro-apoptotic compositions. Thepro-apoptotic compositions used in the methods of the present inventionpreferably induce apoptosis in activated T cells, NKT cells, NK cells,and B cells. The amount and conditions under which the pro-apoptoticcompositions induce desired apoptosis may vary and can be determined bythe skilled artisan using routine optimization. In certain embodiments,a pro-apoptotic composition of the present invention will induceapoptosis without further activation/stimulation. Illustrative examplesof such agents or stimuli include, but are not limited to, deprivationof a growth factor, oxidizing conditions, heat stress, freeze-thawstress, serum starvation, various antibodies, such as anti-CD2,anti-CD3, anti-CD28, anti-CD20, or anti-Fas antibody; MHC-peptidetetramers; Fas ligand, TRAIL, FR901228 (as described in U.S. Pat. No.6,403,555), FK506, annexin, caspases, cytokines such as IL-2 or IL-4,cyclophosphamide, chemotherapeutic agents, V, steroids, corticosteroids,glucocorticoids, rolipram, doxorubicin, chlorambucil, fludarabine,inhibitors of bcl-2, topoisomerase inhibitors, interleukin-1β convertingenzyme (ICE)-binding agents, Shigella IpaB protein, staurosporine,ultraviolet irradiation, gamma irradiation, radiation, tumor necrosisfactor, histone deacetylase inhibitors, and others well known in theart. In certain embodiments, the pro-apoptotic composition comprises asurface, such as a magnetic bead, having attached thereto one or moreagents that binds a cell surface moiety. In this regard, the agent canbe any agent as described herein. In one embodiment, the surface hasattached thereto at least anti-CD3 antibodies. In another embodiment,the surface has attached thereto anti-CD3 and anti-CD28 antibodies. Inaddition, a stimulator of apoptosis can be a polypeptide that is capableof increasing or inducing the apoptotic activity of a cell. Suchpolypeptides include those that directly regulate the apoptotic pathwaysuch as Bax, Bad, Bcl-xL, Bak, Bik, and active caspases as well as thosethat indirectly regulate the pathway. Other illustrative pro-apoptoticcompositions include, but are not limited to, irradiated cells (e.g.donor or recipient (allo) cells), target antigens (e.g. definedautoimmune target antigens such as myelin basic protein (MBP),pancreatic islet cell antigens, cytoplasmic linker protein-170(CLIP-170), Sjogren's syndrome antigen A (SS-A/Ro), Sjogren's syndromeantigen B (SS-B/La), Sjogren's lupus antigen (SL), scleroderma antigen70 (Scl-70)) nucleic acid molecules, proteins or peptides, andnon-protein or non-polynucleotide compounds.

In one aspect of the present invention, one or more pro-apoptoticcompositions is administered to an individual in vivo in conjunctionwith a pharmaceutically acceptable excipient. Any combination ofpro-apoptotic compositions may be administered, such as anti-CD3antibodies, in conjunction with a cytokine such as IL-2 or IL-4,administration of which is described in patent application numberWO9428926. As the skilled artisan will readily recognize, tests on anypro-apoptotic composition used in the methods of the present inventionwould need to be routinely carried out over a range of doses todetermine: 1) the pharmacokinetic behavior of these substances; and 2)safety and identification of any untoward effects 3) optimal doses foreffective induction of apoptosis in cells to be eliminated. This wouldconstitute a Phase I clinical trial. Thus, the particular pro-apoptoticcompositions employed in the methods described herein would requireindividual routine optimization. The pro-apoptotic compositions of thepresent invention can be administered topically, parenterally, or byinhalation. The term “parenteral” includes subcutaneous injections,intravenous, intramuscular, intracisternal injection, or infusiontechniques. These compositions will typically contain an effectiveamount of the pro-apoptotic composition, alone or in combination with aneffective amount of any other active material. Such dosages and desireddrug concentrations contained in the compositions may vary dependingupon many factors, including the intended use, mammal's body weight andage, and route of administration. Preliminary doses can be determinedaccording to animal tests, and the scaling of dosages for humanadministration can be performed according to art-accepted practices.

In one aspect of the present invention, the population of cells isexposed to one or more pro-apoptotic compositions in vitro. As theskilled artisan will readily recognize, tests on any pro-apoptoticcomposition used in the methods of the present invention would need tobe routinely carried out over a range of doses to determine: 1) thebehavior of these substances; and 2) safety and identification of anyuntoward effects 3) optimal doses for effective induction of apoptosisin cells to be eliminated. Thus, the particular pro-apoptoticcompositions employed in the methods described herein would requireindividual routine optimization. In one particular embodiment, thepopulation of remaining cells that has been cleared of unwanted reactivesubpopulations of cells can then be administered to the patient withoutfurther stimulation/activation or expansion.

In one embodiment of the present invention, cells are exposed topro-apoptotic compositions multiple times either alone or in combinationwith other pro-apoptotic compositions. In certain aspects of the presentinvention, it may be preferable to activate/stimulate and in some casesalso expand a mixed population of cells as described below in thesections entitled “Stimulation/Activation of Cell Populations” and“Expansion of Cell Populations” prior to exposure to one or morepro-apoptotic compositions. In one preferred embodiment, the cellsremaining in the population following exposure to a pro-apoptoticcompositions of the present invention, are activated/stimulated andexpanded in vitro as described below in the sections entitled“Stimulation/Activation of Cell Populations” and “Expansion of CellPopulations”. In certain embodiments, the pro-apoptotic composition andthe composition used to activate/stimulate and expand are the samecomposition. In one particular embodiment, a surface having attachedthereto an agent, as described herein, is used as a pro-apoptoticcomposition and further, used to active/stimulate and expand a mixedpopulation. In this regard, certain clonal cells in the population areinduced to undergo apoptosis while others are stumulated/activated andproliferate in response to the composition. In this context, anillustrative composition that can be used both to induce apoptosis in asubpopulation of T cells and to stimulate/activate and expand a mixedpopulation of T cells comprises anti-CD3 and anti-CD28 antibodiesco-immobilized on beads (3×28 beads).

In another embodiment of the present invention, the cells remainingfollowing exposure to one or more pro-apoptotic compositions, arefurther stimulated/activated and expanded in vivo. In vivo stimulationand expansion of the cells of the present invention can be carried outusing any number of cytokines, such as IL-2 and IL-4 or other agentsdescribed herein that simulate cells.

In a further embodiment of the present invention, the cells remainingfollowing exposure to one or more pro-apoptotic compositions, arefurther stimulated/activated and expanded in vitro using the surfacesand agents bound thereto as described below in the sections entitled“Stimulation/Activation of Cell Populations” and “Expansion of T cellPopulations”. The stimulation and activation of the remaining cells thathave not undergone apoptosis using the surfaces of the presentinvention, can increase polyclonality of said remaining population of Tcells as measured by the breadth of the response of the population to agiven antigen. Restoration or increase in polyclonality can be measuredby determining the breadth of response to a particular antigen ofinterest, for example by measuring the number of different epitopesrecognized by antigen-specific cells. This can be carried out usingstandard techniques for generating and cloning antigen-specific T cellsin vitro.

The stimulation and activation using the surfaces of the presentinvention of the remaining cells that have not undergone apoptosis,restores polyclonality to said remaining population of T cells withrespect to expressed TCR genes as indicated by spectratype analysis.Polyclonality of the T cell compositions of the present invention are asdescribed in U.S. Patent Application No. 60/375,733. Spectratypeanalysis is a method for measuring TCR Vβ, Vα, Vγ, or Vδ gene usage by apool of T cells and levels of nucleotide insertion during therecombination process in T cell development (as described in U.S. Pat.No. 5,837,447). Spectratype analysis can be used to measure the breadthor narrowness of the T cell immune response potential. Additionally,spectratype analysis can be used to determine if specific undesiredclonal populations of T cells have been removed from a mixed populationof T cells.

The ability of V, D, and J gene segments to combine together randomlyintroduces a large element of combinatorial diversity into the TCRrepertoire. The precise point at which V, D, and J segments join canvary, giving rise to local amino acid diversity at the junction. Theexact nucleotide position of joining can differ by as much as 10residues resulting in deletion of nucleotides from the ends of the V, D,and J gene segments, thereby producing codon changes at the junctions ofthese segments. Diversity is further increased during the rearrangementprocess when additional nucleotides not encoded by either gene segmentare added at the junction between the joined gene segments. (Thevariability created by this process is called “N-region diversity.”)(Janeway, Travers, Walport. Immunobiology. Fourth Ed., 98 and 150.Elsevier Science Ltd/Garland Publishing. 1999).

The level of diversity for the T cell repertoire can be measured, inpart, by evaluating which TCR Vβ, Vα, Vγ, or Vδ chains are beingemployed by individual T cells within a pool of circulating T cells, andby the number of random nucleotides inserted next to the Vβ gene at theV-D-J or V-J gene junctions. In general, when the circulating T cellpool contains T cells expressing the full range of TCR Vβ, Vα, Vγ, or Vδchains and when those individual V region chains are derived from generecombination events which utilize the broadest array of insertednucleotides, the T cell arm of the immune system will have its greatestpotential for recognizing the universe of potential antigens. When therange of TCR V region chains expressed by the circulating pool of Tcells is limited or reduced, and when expressed TCRs utilize chainsencoded by recombined genes with limited nucleotide insertions, thebreadth of the immune response potential is correspondingly reduced. Theconsequences of this are a reduced ability to respond to the widevariety of antigens leading to increased risks of infection and cancer.

Methods for determining apoptosis are known in the art and aredescribed, for example, in Current Protocols in Immunology, John Wiley &Sons, New York, N.Y., or in U.S. Pat. No. 6,312,684. Illustrative assaysto measure apoptosis comprise DNA ladder, electron or light microscopy,flow cytometry, and different commercially available kits for thedetermination of apoptosis. In certain embodiments, cells are observedfor morphological changes, such as chromatin condensation, cellshrinkage, increased granularity and other indicia of apoptosis known tothose of skill in the art. Chromatin condensation can be detected bystandard methods, such as light microscopy of stained cell preparations.Cell shrinkage and granularity can be readily detected by measuring thelight scattering properties of the cells (Kerr, et al. supra., andWyllie, et al., supra). Observation of single or double strandedfragmentation of DNA into oligonucleosomal ladders often is anotherindication that apoptosis has been induced (Arend, et al., Am. J.Pathol, 136:593, 1990; Wyllie, et al., J. Pathol, 142.:67, 1984).Sometimes, however, apoptotic cells do not exhibit double strandedinternucleosomal DNA fragmentation (Collins, et al., Int. J. Rad. Biol.,62:45 1992; Cohen, et al., Biochem. J., 286:331 1992); instead, singleDNA strand breaks will be observed. Single-strand breaks can readily bedetected using a method of in situ nick end-labeling of the DNA. Thismethod is described by Wyllie, et al. (Br. J. Cancer, 67:20, 1993).

In one embodiment, the cells of the present invention are exposed to agrowth inhibiting composition as described herein. In certainembodiments, the growth inhibiting compositions of the present inventioninhibit growth in at least a substantial portion of at least one clonalpopulation of T cells such that when a mixed population of cells isactivated/stimulated and expanded as described herein, the growthinhibited cells do not expand and are eventually out-competed by themixed population of cells. The end result of this being the effectiveelimination of the growth inhibited cells from the mixed population ofcells.

Exposure to Compositions that Sensitize Cells to FurtherStimulation/Activation

Alternatively, at least a substantial portion of an undesired populationof cells can be eliminated by first sensitizing the cells to furtherstimulation/activation and then further simulating or activating them byexposure to a surface of the present invention. This additionalstimulation/activation induces apoptosis in the sensitized cells,leading to their elimination from the population. The sensitizingcompositions of the present invention also sensitize cells to theeffects of pro-apoptotic compositions described above. Thus, the presentinvention provides for methods to eliminate at least a substantialportion of an undesired clonal population of cells, typically T cells orB cells, from a population of immune cells by exposure to one or morecompositions that sensitize the undesired populations of cells tofurther stimulation/activation or to the effects of a pro-apoptoticcomposition. The present invention further provides for compositionscomprising populations of cells that no longer contain undesired cells,and uses thereof.

In one embodiment, exposure to a composition that sensitizes to furtheractivation or stimulation occurs naturally in vivo, such as in thesetting of autoimmune diseases. In this regard, auoimmune cells areexposed to autoantigen in vivo and are thus sensitized. Upon furtherstimulation/activation, such as by using the methods of the presentinvention using a surface as described herein (e.g., a surface havingattached thereto one or more agents that ligate a cell surface moiety,such as anti-CD3 and anti-CD28 antibodies), these autoimmune cells areinduced to undergo apoptosis.

In one aspect of the present invention, a population of immune cells isexposed to a composition or compositions that sensitize to furtheractivation or stimulation, at least a portion of cells, e.g. previouslyhighly activated T cells or B cells. In a preferred embodiment, thesensitized cells comprise undesired autoreactive T or B cells (e.g., inthe setting of multiple sclerosis, such sensitized cells would compriseMBP-specific T cells that are sensitized in vivo as a results of theaberrant immune regulation associated with this disease). In a furtherembodiment, the sensitized cells comprise alloreactive cells present indonor hematopoietic stem cell. In yet a further embodiment, thesensitized cells comprise alloreactive cells from a potential organtransplant recipient.

In one embodiment of the present invention, the sensitizing compositioncomprises irradiated cells. In a particular embodiment, the irradiatedcells are from a hematopoietic stem cell transplant recipient and thecells to be sensitized are from the hematopoietic stem cell transplantdonor. In another embodiment, the sensitizing composition comprisesirradiated cells from an organ donor and the cells to be sensitized arecells from the organ recipient. In certain embodiments, the cells to besensitized are cells from an organ recipient post-transplant. Cells aretypically irradiated with gamma rays in the range of about 3000 to 3600rads, more preferably at about 3300 rads. Other irradiated cells thatmay be useful in the present invention, such as lymphoblastoid or tumorcell lines are typically irradiated with gamma rays in the range ofabout 6000 to 10,000 rads, more preferably at about 8000 rads. Cells mayalso be treated by other means such as with chemical agents (e.g.,etiposide, mitomycin, and the like).

Sensitizing compositions of the present invention include anycomposition or combination of compositions that sensitizes immune cells,such as T, NKT, NK, or B cells, to subsequent stimulation such thatsubsequent stimulation or activation induces apoptosis. Sensitizingcompositions of the present invention also include any composition orcombination of compositions that sensitizes immune cells, such as T or Bcells, to subsequent exposure to a pro-apoptotic composition.Sensitizing compositions of the present invention include but are notlimited to antibodies such as anti-CD2, anti-CD3, anti-FAS; MHC-peptidedimers or tetramers, cytokines such as IL-2, TRAIL, compounds such asrolipram, doxorubicin, chlorambucil and fludarabine. Sensitizingcompositions also include FAS-ligand and the natural ligands for CD2 andCD3. Sensitizing compositions also include inhibitors of bcl-2, such asthose described in U.S. Pat. No. 6,277,844, topoisomerase inhibitors,such as etoposide, CPT-11 and topotecan, and others, such as describedin U.S. Pat. No. 5,834,012. Other illustrative sensitizing compositionsinclude interleukin-1β converting enzyme (ICE)-binding agents thatinduce apoptosis, such as Shigella IpaB protein described in U.S. Pat.No. 5,972,899, or compounds described in U.S. Pat. Nos. 6,350,741,6,294,546 and 6,329,365.

Illustrative sensitizing compositions of the present invention alsocomprise autoantigens. Autoantigens may be defined autoimmune targetantigens e.g. defined autoimmune target antigens for example, inmultiple sclerosis, the target antigen identified as myelin basicprotein (MBP) MBP 84-102, or MBP 143-168; pancreatic islet cellantigens; in uveitis, the S Antigen; or in rheumatoid arthritis, type IIor other types of collagen; in SLE, cytoplasmic linker protein-170(CLIP-170); Sjogren's syndrome antigen A (SS-A/Ro), Sjogren's syndromeantigen B (SS-B/La), Sjogren's lupus antigen (SL); scleroderma antigen70 (Scl-70); in Grave's disease, thyroid receptor; in Myasthena gravis,acetylcholine receptor, nucleic acid molecules, proteins or peptides,and non-protein or non-polynucleotide compounds. Autoantigens of thepresent invention also comprise peptide mixtures eluted from MHCmolecules known to be associated with autoimmunity, for example, HLA-DQand -DR molecules that confer susceptibility to several commonautoimmune diseases, such as type 1 diabetes, rheumatoid arthritis andmultiple sclerosis, or HLA-B27 molecules known to confer susceptibilityto reactive arthritis and ankylosing spondylitis. Autoantigens of thepresent invention may also be synthesized peptides predicted to bind toMHC molecules associated with autoimmune diseases. It should be notedthat sensitization may occur naturally as a process of autoimmunedisease. As such, the “pre sensitized” autoreactive(autoantigen-specific) T cells exist in patients and can be eliminatedor otherwise substantially reduced directly through the XCELLERATE™process as described herein.

The present invention further provides sensitizing compositions forselectively eliminating at least a substantial portion of a populationof T cells expressing a specific Vβ, Vα, Vγ, or Vδ gene. For example,antibodies specific for a particular Vβ, Vα, Vγ, or Vδ gene can be usedto specifically sensitize the T cells according to the methods of thepresent invention. Alternatively, T cells expressing a particular Vβ,Vα, Vγ, or Vδ gene of interest can be negatively selected, therebyeliminating at least a substantial portion of them from a population.

In further embodiments of the present invention, one or more sensitizingcompositions are used simultaneously and for times sufficient to inducethe desired sensitization.

As described above with pro-apoptotic compositions, the presentinvention provides for methods wherein compositions that sensitize cellsto further stimulation/activation or the effects of pro-apoptoticcompositions, are administered in vivo or in vitro, or a combination ofthe two. As with any medicinal substance, or biologic, tests on anyagents that sensitize cells to further stimulation/activation to beadministered in vivo, such as numerous pro-apoptotic compounds,antibodies, peptides and proteins used for immunization would need to beroutinely carried out over a range of doses to determine: 1) thepharmacokinetic behavior of these substances; 2) their immunogenicity;and 3) safety and identification of any untoward effects. This wouldconstitute a Phase I clinical trial. Thus, the particular agents thatsensitize cells to further stimulation/activation employed in themethods of the present invention (for example, in multiple sclerosis,the target antigen identified as MBP 84-102, or MBP 143-168; in uveitis,the S Antigen; or in rheumatoid arthritis, type II collagen) wouldrequire individual routine optimization. The sensitizing compositions ofthe present invention can be administered topically, parenterally, or byinhalation. The term “parenteral” includes subcutaneous injections,intravenous, intramuscular, intracisternal injection, or infusiontechniques. These compositions will typically contain an effectiveamount of the sensitizing composition, alone or in combination with aneffective amount of any other active material. Such dosages and desireddrug concentrations contained in the compositions may vary dependingupon many factors, including the intended use, mammal's body weight andage, and route of administration. Preliminary doses can be determinedaccording to animal tests, and the scaling of dosages for humanadministration can be performed according to art-accepted practices.

Ample evidence from the development of vaccines suggests that eithersynthetic peptides or recombinant DNA-derived proteins are effective ineliciting an immune response in humans. These studies also provideguidance as to the range of doses effective for immunization (Zajoc, B.A., D. J. West, W. J. McAleer and E. M. Scolnick, Overview of clinicalstudies with Hepatitis B vaccine made by recombinant DNA, J. Infect.13:(Suppl A)39-45 (1986). Yamamoto, S., T. Kuroki, K. Kurai and S. Iino,Comparison of results for phase I studies with recombinant andplasma-derived hepatitis B vaccines, and controlled study comparingintramuscular and subcutaneous injections of recombinant hepatitis Bvaccine, J. Infect. 13:(Suppl A)53-60 (1986). Francis, D. P. et al., Theprevention of Hepatitis B with vaccine, Ann. Int. Med. 97:362-366(1982). Putney et al., Features of HIV envelope and development of asubunit vaccine, AIDS Vaccine Research and Clinical Trials, S. Putneyand B. Bolognesi, eds. (New York: Dekker) pp. 3-62 (1990). Steven, V. C.and W. R. Jones, Vaccines to prevent pregnancy, New Generation Vaccines,G. C. Woodrow and M. M. Levine, eds. (New York: Dekker) pp. 879-900(1990). Herrington et al., Safety and immunogenicity in man of asynthetic peptide malaria vaccine against Plasmodium Falcipariumsporozoites, Nature, 328:257-259 (1987)).

In one embodiment of the present invention, immunization (i.e. in vivosensitization) with an agent that sensitizes cells to furtherstimulation/activation or exposure to a pro-apoptotic composition, isthen followed by a waiting period during which the agent activates thesubset of cells bearing reactive receptors, such as T cells bearingreactive TCRs or B cells expressing specific antibody receptors, causingthem to express cytokine receptors, such as the IL-2 receptor. Forexample, this process will induce IL-2 receptors only on T cells thathave been antigenically-stimulated. Based on studies of both human andmouse T cells in vitro, between about 12 to about 24 hours after antigenexposure are required to express significant numbers of IL-2 receptors,and as long as about 72 hours are required to express optimal numbers ofIL-2 receptors on the majority of T cells. Thus, the waiting period canbe as short as about 12 hours or as long as about 72 hours, and in thecase of various disease states, due to retarded immune responsiveness,this period may be as long as 120 hours, becoming increasingly optimaltoward the upper end of this range.

In one embodiment of the present invention, IL-2, or other appropriatecytokines, such as IL-4, are administered to the patient to induceapoptosis in the activated cells as described above. Administration ofIL-2 to humans has been well-studied in cancer patients, and variousdoses have been evaluated (Loize, M. T., L. W. Frana, S. O. Sharrow, R.J. Robb and S. A. Rosenberg, In vivo administration of purified humaninterleukin 2. I. Half-life and immunologic effects of the Jurkat cellline-derived interleukin 2. J. Immunol. 134:157-166 (1985). Lotze, J.T., Y. L. Malory, S. E. Ettinghausen, A. A. Rayner, S. O. Sharrow, C. A.Y. Seipp, M. C. Custer and S. A. Rosenberg, In vivo administration ofpurified human interleukin 2. II. Half-life, immunologic effects, andexpansion of peripheral lymphoid cells in vivo with recombinant IL 2. J.Immunol. 135:2865-2875 (1985). Donahue, J. H. and S. A. Rosenberg, Thefate of interleukin-2 after in vivo administration, J. Immunol.130:2203-2208 (1983). Belldegrun, A., M. M. Muul and S. A Rosenberg,Interleukin 2 expanded tumor-infiltrating lymphocytes in human renalcell cancer: isolation, characterization, and antitumor activity, CancerResearch 48:206-214 (1988). Rosenberg, S. A., M. T. Lotze, L. M. Muul,S. Leitman, A. E. Chang, S. E. Ettinghausen, Y. L. Malory, J. M.Skibber, E. Shiloni, J. T. Vetto, C. A. Seipp, C. Simpson and C. M.Reichert, Observations on the systemic administration of autologouslymphokine-activated killer cells and recombinant interleukin-2 topatients with metastatic cancer, New Eng. J. Med. 313:1485-1492(1985).). Data indicate that IL-2 should be given I.V., either asfrequent bolus doses or as a continuous infusion. Doses that have beenpreviously established range between about 300 to about 3000units/kg/hour continuous infusion, or from 104 to 106 units/kg I.V.bolus.

In one aspect of the present invention, the population of cells isexposed to one or more sensitizing compositions in vitro. As the skilledartisan will readily recognize, tests on any sensitizing compositionused in the methods of the present invention would need to be routinelycarried out over a range of doses to determine: 1) the pharmacokineticbehavior of these substances; and 2) safety and identification of anyuntoward effects 3) optimal doses for effective induction of apoptosisin cells to be eliminated. Thus, the particular sensitizing compositionsemployed in the methods described herein would require individualroutine optimization.

In one embodiment of the present invention, cells are collected from anindividual previously treated in vivo with an agent that sensitizescells to further stimulation/activation. Cells are then furtherstimulated/activated to induce apoptosis and then expanded in vitro asdescribed below.

Stimulation of Sensitized Cells to Induce Apoptosis of Cells to beEliminated From a Mixed Population of Cells

In one aspect of the present invention, a population of immune cellscomprising sensitized cells as described above is further activated orstimulated to induce apoptosis as described below in the sectionentitled “Stimulation/Activation of Cell Populations”, therebyeliminating the sensitized cells, such as autoreactive or alloreactiveT- or B-cells, from the mixed population of cells. At the same time, thedesired cells that remain, e.g., those cells that are not sensitized toundergo apoptosis, are activated and stimulated to expand, therebyresulting in a population of activated cells from which at least asubstantial portion of unwanted subpopulations of T (or B cells) havebeen eliminated. As mentioned previously, stimulation/activation asdescribed herein may be carried out on cells remaining followingexposure of a mixed population of cells directly to pro-apoptoticcompositions. Furthermore, the subsequent stimulation and activationprovided by the present invention restores polyclonality to thepopulation of T cells with respect to expressed TCR genes as indicatedby spectratype analysis.

In one embodiment of the present invention, sensitized cells arestimulated/activated as described below multiple times with or withoutadditional sensitizing composition, as many times as is necessary toeliminate at least a substantial portion of the undesired cells. Forexample in the setting of an autoimmune disease, the present inventionprovides for methods to stimulate cells a second or more times in thepresence of antigen (i.e., sensitizing composition) after the initialround of stimulation/activation. Likewise, in the setting ofhematopoietic stem cell transplantation, the present invention providesfor methods to stimulate cells from the hematopoietic stem cell donor asecond or more times, or as many times as necessary to eliminate atleast a substantial portion of the undesired cells, in the presence ofirradiated cells from a hematopoietic stem cell transplant recipient. Inthe setting of organ transplantation, the present invention provides formethods to stimulate cells from the organ recipient a second or moretimes, or as many times as is necessary to eliminate at least asubstantial portion of undesired cells, if necessary in the presence ofirradiated cells from the organ donor. In one embodiment, the methods ofthe present invention are carried out on cells from a patient (e.g. hostcells) post-transplant in order to eliminate undesired cells. In certainembodiments, it may not be necessary to eliminate all of the undesiredcells, for example in the setting of hematopoietic stem celltransplantation for certain types of cancer, graft versus leukemic celleffect may be desired.

In certain aspects of the present invention, it may be preferable tostimulate/activate and in some cases expand a mixed population of cellsas described below in the sections entitled “Stimulation/Activation ofCell Populations” and “Expansion of Cell Populations” prior to exposureto one or more agents that sensitize cells to furtherstimulation/activation and subsequent stimulation.

In further aspects of the present invention, the cells are sensitizedand then exposed to a pro-apoptotic composition, thereby eliminating atleast a substantial portion of cells that have become sensitized to theeffects of the pro-apoptotic composition. The cells remaining in thepopulation can then be further stimulated/activated and expanded asdescribed below.

In one embodiment, the cells of the present invention are exposed to agrowth inhibiting composition as described herein. In certainembodiments, the growth inhibiting compositions of the present inventioninhibit growth in at least a substantial portion of at least one clonalpopulation of T cells such that when a mixed population of cells isactivated/stimulated and expanded as described herein, the growthinhibited cells do not expand and are eventually out-competed by themixed population of cells. The end result of this being the effectiveelimination of the growth inhibited cells from the mixed population ofcells.

Generation of a Substantially Pure CD3⁺ CD28⁺ T Cell Population

The present invention provides methods for the generation of asubstantially pure population of CD3⁺ CD28⁺ T cells from a population ofimmune cells. For the purposes of the present invention, a population ofsubstantially pure CD3⁺ CD28⁺ T cells contains less than 10% CD3⁺ CD28⁻T cells. In certain embodiments, a population of substantially pure CD3⁺CD28⁺ T cells contains less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%,0.5%, or 0.1% CD3⁺ CD28⁻ T cells.

A pure population of CD3⁺ CD28⁺ T cells can be generated by magneticconcentration, selection, and stimulating the mixed population of Tcells with a composition capable of stimulating both CD3 and CD28molecules on the surface of a T cell. Selection and stimulation of boththe CD3 and CD28 molecules on the surface of a cell results in theactivation and proliferation of this subset of cells. Conversely, underconditions described herein, exposure of a CD3⁺ CD28⁻ T cell to acomposition capable of selecting and stimulating both CD3 and CD28surface molecules would be insufficient to induce both activation andexpansion of this population of T cells. Further, shortening incubationtime with CD3/CD28 beads as described herein, favors selection of CD3⁺CD28⁺ cells at the expense of CD3⁺ CD28⁻ cells (e.g., a 15 minuteselection at 1 r.p.m. at room temperature, followed by magneticconcentration leaves many or most CD3⁺ CD28⁻ cells behind.) Thus, in oneembodiment, a short incubation with a surface as described hereinfollowed by a short magnetic selection is used to preferentially selector enrich for CD28⁺T cells while leaving CD28⁻ cells behind. Further thetemperature of incubation, the rate of mixing during the incubation, andthe exposure to the magnetic field, can all be varied to preferentiallyselect for CD28⁺cells. In certain embodiments, antibodies other thananti-CD28 or in conjuction with anti-CD28 can be used, for exampleanti-NKG2D antibody.

Triggering of the TCR by either a specific antigen or by a moleculecapable of stimulating the CD3 surface molecule, for example an anti-CD3antibody, is considered insufficient to induce expansion and lymphokinesecretion unless supplemented by co-stimulatory signals, i.e., thespecific stimulation of the CD28 molecule. In fact, in the absence ofco-stimulation, these T cells may acquire a state of non-responsivenessor anergy.

Thus, the methods of the present invention, e.g., the stimulation andselection of a mixed population of T cells using a composition capableof triggering CD3 and simulating CD28, would result in the generation ofa substantially pure population of CD3⁺ CD28⁺ T cells.

In certain embodiments, it may be desirable to use the CD28⁻ populationof T cells. Without being bound by theory, this population may contain Tcells, such as tumor-specific, or virus-specific T cells of interestthat could be enriched and used for therapy as described herein, eitheras is or following further culture/expansion.

In one embodiment of the present invention, this population ofsubstantially pure CD3⁺ CD28⁺ T cells can be used to treat acute orchronic GVHD. In other embodiments, a population of substantially pureCD3⁺ CD28⁺ T cells can be used to treat autoimmune diseases, such asrheumatoid arthritis, multiple sclerosis, insulin dependent diabetes,Addison's disease, celiac disease, chronic fatigue syndrome, colitis,Crohn's disease, fibromyalgia, lupus, psoriasis, Sjogren's syndrome,hyperthyroidism/Graves disease, hypothyroidism/Hashimoto's disease,insulin-dependent diabetes (type 1), Myasthenia Gravis, endometriosis,scleroderma, pernicious anemia, Goodpasture syndrome, Wegener's diseaseand rheumatic fever. In a further embodiment, the cells of the presentinvention can be used to treat autoimmunity associated with largegranular lymphocyte leukemia (LGL). A mixed population of immune cellscould be removed from a donor and these cells stimulated with acomposition capable of stimulating CD3 and CD28 molecules. While notwanting to be bound by theory, it is postulated that this stimulationresults in the specific activation and expansion of CD3⁺ CD28⁺ T cells,and result in the anergy of T cells that lack the expression of theco-stimulatory molecule, CD28. Once the pure population of CD3⁺ CD28⁺ Tcells has been generated, these cells can then be infused for thetreatment of an autoimmune disease, LGL, or GVHD.

Stimulation/Activation of Cell Populations

The stimulated and activated T cells of the present invention aregenerated by cell surface moiety ligation that induces activation. Incertain embodiments, the stimulated and activated T cells are generatedby activating a population of T cells solely via engagement of the TCR,for example using anti-TCR antibodies, anti-CD3 antibodies, or naturalligands for the TCR. In certain embodiments, the stimulated andactivated T cells are generated by activating a population of T cellsand stimulating an accessory molecule on the surface of the T cells witha ligand which binds the accessory molecule, as described for example,in U.S. patent application Ser. Nos. 08/253,694, 08/435,816, 08/592,711,09/183,055, 09/350,202, 09/252,150, 10/133,236, 10/187,467, 10/350,305,published PCT application WO03024989, and patent numbers 6,352,694,5,858,358 and 5,883,223. In the context of sensitized cells describedabove, activating said sensitized population of T cells and stimulatingan accessory molecule on the surface of said sensitized T cells with aligand which binds the accessory molecule induces apoptosis andsubsequent elimination of the cells.

Generally, T cell activation of cells may be accomplished by cellsurface moiety ligation, such as stimulating the T cell receptor(TCR)/CD3 complex or the CD2 surface protein. A number of anti-human CD3monoclonal antibodies are commercially available, exemplary are, cloneBC3 (XR-CD3; Fred Hutchinson Cancer Research Center, Seattle, Wash.),OKT3, prepared from hybridoma cells obtained from the American TypeCulture Collection, and monoclonal antibody G19-4. Similarly,stimulatory forms of anti-CD2 antibodies are known and available.Stimulation through CD2 with anti-CD2 antibodies is typicallyaccomplished using a combination of at least two different anti-CD2antibodies. Stimulatory combinations of anti-CD2 antibodies that havebeen described include the following: the T11.3 antibody in combinationwith the T11.1 or T11.2 antibody (Meuer et al., Cell 36:897-906, 1984),and the 9.6 antibody (which recognizes the same epitope as T11.1) incombination with the 9-1 antibody (Yang et al., J. Immunol.137.1097-1100, 1986). Other antibodies that bind to the same epitopes asany of the above described antibodies can also be used. Additionalantibodies, or combinations of antibodies, can be prepared andidentified by standard techniques. Stimulation may also be achievedthrough contact with superantigens (e.g., Staphylococcus enterotoxin A(SEA), Staphylococcus enterotoxin B (SEB), Toxic Shock Syndrome Toxin 1(TSST-1)), endotoxin, or through a variety of mitogens, including butnot limited to, phytohemagglutinin (PHA), phorbol myristate acetate(PMA) and ionomycin, lipopolysaccharide (LPS), T cell mitogen, and IL-2.

To further activate a population of T cells, a co-stimulatory oraccessory molecule on the surface of the T cells, such as CD28, isstimulated with a ligand that binds the accessory molecule. Accordingly,one of ordinary skill in the art will recognize that any agent,including an anti-CD28 antibody or fragment thereof capable ofcross-linking the CD28 molecule, or a natural ligand for CD28 can beused to stimulate T cells. Exemplary anti-CD28 antibodies or fragmentsthereof useful in the context of the present invention includemonoclonal antibody 9.3 (IgG2_(a)) (Bristol-Myers Squibb, Princeton,N.J.), monoclonal antibody KOLT-2 (IgG1), 15E8 (IgG1), 248.23.2 (IgM),clone B-T3 (XR-CD28; Diaclone, Besançon, France) and EX5.3D10 (IgG2_(a))(ATCC HB11373). Exemplary natural ligands include the B7 family ofproteins, such as B7-1 (CD80) and B7-2 (C D86) (Freedman et al., J.Immunol. 137:3260-3267, 1987; Freeman et al., J. Immunol. 143:2714-2722,1989; Freeman et al., J. Exp. Med. 174:625-631, 1991; Freeman et al.,Science 262:909-911, 1993; Azuma et al., Nature 366:76-79, 1993; Freemanet al., J. Exp. Med. 178:2185-2192, 1993).

Other illustrative accessory molecules on the surface of the T cellsthat can be stimulated with a ligand that binds the accessory moleculein the present invention include, but are not limited to, NKG2D, CD54,LFA-1, ICOS, and CD40.

In addition, binding homologues of a natural ligand, whether native orsynthesized by chemical or recombinant techniques, can also be used inaccordance with the present invention. Other agents may include naturaland synthetic ligands. Agents may include, but are not limited to, otherantibodies or fragments thereof, growth factor, cytokine, chemokine,soluble receptor, steroid, hormone, mitogen, such as PHA, or othersuperantigens.

As described earlier, the subsequent stimulation and activation of theremaining cells that have not undergone apoptosis or have not beensensitized to undergo apoptosis, restores polyclonality to saidremaining population of T cells with respect to expressed TCR genes asindicated by spectratype analysis.

Expansion of Cell Populations

Generally, the present invention provides for expansion of thepopulation of cells that remains following exposure of the population toa pro-apoptotic compositions or a sensitizing composition and anysubsequent induction of apoptosis in undesired subpopulations of cells,preferably autoreactive or undesired alloreactive T cells. In oneembodiment of the invention, the remaining T cells may be stimulated bya single agent. In another embodiment, remaining T cells are stimulatedwith two or more agents, one that induces a primary signal andadditional agents that induce one or more co-stimulatory signals.Ligands useful for stimulating a single signal or stimulating a primarysignal and an accessory molecule that stimulates a second signal may beused in soluble form, attached to the surface of a cell, or immobilizedon a surface as described herein. A ligand or agent that is attached toa surface serves as a “surrogate” antigen presenting cell (APC). In apreferred embodiment both primary and secondary agents areco-immobilized on a surface. In one embodiment, the molecule providingthe primary activation signal, such as a CD3 ligand, and theco-stimulatory molecule, such as a CD28 ligand, are coupled to the samesurface, for example, a particle. Further, as noted earlier, one, two,or more stimulatory molecules may be used on the same or differingsurfaces.

The cell population may be stimulated as described herein, such as bycontact with an anti-CD3 antibody or an anti-CD2 antibody immobilized ona surface, or by contact with a protein kinase C activator (e.g.,bryostatin) in conjunction with a calcium ionophore. For co-stimulationof an accessory molecule on the surface of the T cells, a ligand thatbinds the accessory molecule is used. For example, a population of CD4⁺cells can be contacted with an anti-CD3 antibody and an anti-CD28antibody, under conditions appropriate for stimulating proliferation ofthe T cells. Alternatively, a population of cells can be contacted withPMA and ionomycin. Similarly, to stimulate proliferation of CD8⁺ Tcells, an anti-CD3 antibody and the anti-CD28 antibody B-T3, XR-CD28(Diaclone, Besançon, France) can be used as can other methods commonlyknown in the art (Berg et al., Transplant Proc. 30(8):3975-3977, 1998;Haanen et al., J. Exp. Med. 190(9):1319-1328, 1999; Garland et al., JImmunol Meth. 227(1-2):53-63, 1999).

The primary stimulatory signal and the co-stimulatory signal for the Tcell may be provided by different protocols. For example, the agentsproviding each signal may be in solution or coupled to a surface. Whencoupled to a surface, the agents may be coupled to the same surface(i.e., in “cis” formation) or to separate surfaces (i.e., in “trans”formation). Alternatively, one agent may be coupled to a surface and theother agent in solution. In one embodiment, the agent providing theco-stimulatory signal is bound to a cell surface and the agent providingthe primary activation signal is in solution or coupled to a surface. Incertain embodiments, both agents can be in solution. In anotherembodiment, the agents may be in soluble form, and then cross-linked toa surface, such as a cell expressing Fc or Sc receptors or an antibodyor other binding agent which will bind to the agents. In a preferredembodiment, the two agents are immobilized on a spherical orsemi-spherical surface, the prototypic examples being beads or cells,either on the same bead, i.e., “cis,” or to separate beads, i.e.,“trans.” By way of example, the agent providing the primary activationsignal is an anti-CD3 antibody and the agent providing theco-stimulatory signal is an anti-CD28 antibody; and both agents areco-immobilized to the same bead in equivalent molecular amounts. In oneembodiment, a 1:1 ratio of each antibody bound to the beads for T cellexpansion and T cell growth is used. In certain aspects of the presentinvention, a ratio of anti CD3:anti-CD28 antibodies (CD3:CD28) bound tothe beads is used such that an increase in T cell expansion is observedas compared to the expansion observed using a ratio of 1:1. In oneparticular embodiment an increase of from about 0.5 to about 3 fold isobserved as compared to the expansion observed using a ratio of 1:1. Inone embodiment, the ratio of anti-CD3:anti-CD28 (CD3:CD28) antibodybound to the beads ranges from about 100:1 to 1:100 and all integervalues there between. In certain embodiments, the ratio of CD3:CD28 isat least about 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 65:1, 60:1, 55:1,50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 15:1, 10:1, 9:1, 8:1, 7:1,6:1, 5:1, 4:1, 3:1, 2:1, or 1:1. In one aspect of the present invention,more anti-CD28 antibody is bound to the particles than anti-CD3antibody, i.e. the ratio of CD3:CD28 is less than one. In certainembodiments of the invention, the ratio of anti-CD28 antibody to antiCD3 antibody bound to the beads is greater than 2:1. In one particularembodiment, a 1:200 CD3:CD28 ratio of antibody bound to beads is used.In one particular embodiment, a 1:150 CD3:CD28 ratio of antibody boundto beads is used. In one particular embodiment, a 1:100 CD3:CD28 ratioof antibody bound to beads is used. In another embodiment, a 1:75CD3:CD28 ratio of antibody bound to beads is used. In a furtherembodiment, a 1:50 CD3:CD28 ratio of antibody bound to beads is used. Inanother embodiment, a 1:45 CD3:CD28 ratio of antibody bound to beads isused. In another embodiment, a 1:40 CD3:CD28 ratio of antibody bound tobeads is used. In another embodiment, a 1:35 CD3:CD28 ratio of antibodybound to beads is used. In another embodiment, a 1:30 CD3:CD28 ratio ofantibody bound to beads is used. In another embodiment, a 1:25 CD3:CD28ratio of antibody bound to beads is used. In another embodiment, a 1:20CD3:CD28 ratio of antibody bound to beads is used. In anotherembodiment, a 1:15 CD3:CD28 ratio of antibody bound to beads is used. Inone preferred embodiment, a 1:10 CD3:CD28 ratio of antibody bound tobeads is used. In another embodiment, a 1:5 CD3:CD28 ratio of antibodybound to beads is used. In another embodiment, a 1:4 CD3:CD28 ratio ofantibody bound to beads is used. In another embodiment, a 1:3 CD3:CD28ratio of antibody bound to the beads is used. In yet another embodiment,a 3:1 CD3:CD28 ratio of antibody bound to the beads is used.

Ratios of particles to cells from 1:500 to 500:1 and any integer valuesin between may be used to stimulate T cells or other target cells. Asthose of ordinary skill in the art can readily appreciate, the ratio ofparticle to cells may depend on particle size relative to the targetcell. For example, small sized beads could only bind a few cells, whilelarger beads could bind many. In certain embodiments the ratio of cellsto particles ranges from 1:100 to 100:1 and any integer valuesin-between and in further embodiments the ratio comprises 1:50 to 50:1and any integer values in between. In another embodiment, the ratio ofcells to particles ranges from 1:9 to 9:1 and any integer values inbetween, can also be used to stimulate T cells. The ratio of anti-CD3-and anti-CD28-coupled particles to T cells that result in T cellstimulation can vary as noted above, however certain preferred valuesinclude at least 1:150, 1:125, 1:100, 1:75, 1:50, 1:40, 1:30, 1:20,1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2.5, 1:2, 1:1, 2:1, 3:1, 4:1,5:1, 6:1, 7:1, 8:1, 9:1, 10:1, and 15:1, with one preferred ratio beingat least 1:1 particles per T cell. In one particular embodiment, thepreferred ratio of particles to cells is 1:5 or 1:10. In one embodiment,a ratio of particles to cells of 1:1 or less is used.

In further embodiments, the ratio of particles to cells can be varieddepending on the day of stimulation. For example, in one embodiment, theratio of particles to cells is from 1:1 to 10:1 on the first day andadditional particles are added to the cells every day or every other daythereafter for up to 10 days, at final ratios of from 1:1 to 1:10 (basedon cell counts on the day of addition). In another embodiment, the ratioof particles to cells is at least about 1:2.5 on the first day andadditional particles are added to the cells on day 5 at about 1:10,1:25, 1:50 or 1:100, on day 7 at 1:10, 1:25, 1:50, or 1:100 and on day 9at 1:10, 1:25, 1:50, or 1:100. In one particular embodiment, the ratioof particles to cells is 1:1 on the first day of stimulation andadjusted to 1:5 on the third and fifth days of stimulation. In anotherembodiment, particles are added on a daily or every other day basis to afinal ratio of 1:1 on the first day, and 1:5 on the third and fifth daysof stimulation. In another embodiment, the ratio of particles to cellsis 2:1 on the first day of stimulation and adjusted to 1:10 on the thirdand fifth days of stimulation. In another embodiment, particles areadded on a daily or every other day basis to a final ratio of 1:1 on thefirst day, and 1:10 on the third and fifth days of stimulation. One ofskill in the art will appreciate that a variety of other ratios may besuitable for use in the present invention. In particular, ratios willvary depending on particle size and on cell size and type.

One aspect of the present invention stems from the surprising findingthat using different bead:cell ratios can lead to different outcomeswith respect to expansion of antigen-specific T cells. In particular,bead:cell ratios can be varied to selectively expand or deleteantigen-specific (memory) T cells. In one embodiment, the particularbead:cell ratio used selectively deletes antigen-specific T cells.Specifically, high bead:cell ratios, such as about 5:1, 10:1, 15:1,20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, and higher, induce deletion ofantigen-specific T cells. Without being bound by theory, it is thoughtthat the antigen-specific T cells are sensitized to further stimulation.Thus, the key appears to be the strength of the T cell activationsignal: selective expansion of memory T cells (antigen-specific T cells)occurs with “weak” signals while selective deletion of memory T cellsoccurs with “strong” signals. The quantity of the CD3/TCR (and CD28)receptors that bound by ligands determines the signal strength. Thus,stimulation with high bead:cell ratios provides a high concentration ofstimulating antibody (i.e., “strong” signal), leading toover-stimulation of antigen-specific T cells, causing them to die,either by apoptosis or other mechanisms. Thus, in this regard, the beadcompositions described herein are functioning as a pro-apoptoticcomposition. Further, in this regard, as the skilled artisan wouldappreciate, in certain embodiments, the same composition used as apro-apoptotic composition (e.g., a surface having attached thereto anagent that stimulates a cell surface moiety, such as the beadcompositions described herein) is used to expand the remaining mixedpopulation of cells for use in any variety of immunotherapeutic settingsas described herein. Using lower bead:cell ratios provides a stimulationsignal to antigen-specific T cells that does not over-stimulate, butrather induces rapid proliferation of these cells. In a furtherembodiment, the particular bead:cell ratio used selectively expandsantigen-specific T cells. The skilled artisan would readily appreciatethat any ratio can be used as long as the desired expansion or deletionoccurs. Therefore, the compositions and methods described herein can beused to expand specific populations of T cells, or to delete specificpopulations of T cells, for use in any variety of immunotherapeuticsettings described herein.

Using certain methodologies it may be advantageous to maintain long-termstimulation of a population of T cells following the initial activationand stimulation, by separating the T cells from the stimulus after aperiod of about 7 to about 14 days. The rate of T cell proliferation ismonitored periodically (e.g., daily) by, for example, examining the sizeor measuring the volume of the T cells, such as with a Coulter Counter.In this regard, a resting T cell has a mean diameter of about 6.8microns, and upon initial activation and stimulation, in the presence ofthe stimulating ligand, the T cell mean diameter will increase to over12 microns by day 4 and begin to decrease by about day 6. When the meanT cell diameter decreases to approximately 8 microns, the T cells may bereactivated and re-stimulated to induce further proliferation of the Tcells. Alternatively, the rate of T cell proliferation and time for Tcell re-stimulation can be monitored by assaying for the presence ofcell surface molecules, such as, CD154, CD54, CD25, CD137, CD134, whichare induced on activated T cells.

For inducing long-term stimulation of a population of CD4⁺ and/or CD8⁺ Tcells, it may be necessary to reactivate and re-stimulate the T cellswith a stimulatory agent such as an anti-CD3 antibody and an anti-CD28antibody (e.g. B-T3, XR-CD28 (Diaclone, Besançon, France)) several timesto produce a population of CD4⁺ or CD8⁺ cells increased in number fromabout 10 to about 1,000-fold the original T cell population. Forexample, in one embodiment of the present invention, T cells arestimulated as described for 2-3 times. In further embodiments, T cellsare stimulated as described for 4 or 5 times. Using the presentmethodology, it is possible to achieve T cell numbers from about 100 toabout 100,000-fold that have increased polyclonality as compared toprior to stimulation. Moreover, T cells expanded by the method of thepresent invention secrete substantial levels of cytokines (e.g., IL-2,IFN-γ, IL-4, GM-CSF and TNF-α) into the culture supernatants. Forexample, as compared to stimulation with IL-2, CD4⁺ T cells expanded byuse of anti-CD3 and anti-CD28 co-stimulation secrete high levels ofGM-CSF and TNF-α into the culture medium. These cytokines can bepurified from the culture supernatants or the supernatants can be useddirectly for maintaining cells in culture. Similarly, the T cellsexpanded by the method of the present invention together with theculture supernatant and cytokines can be administered to support thegrowth of cells in vivo.

In one embodiment, T cell stimulation is performed, for example withanti-CD3 and anti-CD28 antibodies co-immobilized on beads (3×28 beads),for a period of time sufficient for the cells to return to a quiescentstate (low or no proliferation) (approximately 8-14 days after initialstimulation). The stimulation signal is then removed from the cells andthe cells are washed and infused back into the patient. The cells at theend of the stimulation phase are rendered “super-inducible” by themethods of the present invention, as demonstrated by their ability torespond to antigens and the ability of these cells to demonstrate amemory-like phenotype, as is evidence by the examples. Accordingly, uponre-stimulation either exogenously or by an antigen in vivo afterinfusion, the activated T cells demonstrate a robust responsecharacterized by unique phenotypic properties, such as sustained CD154expression, increased cytokine production, etc.

In further embodiments of the present invention, the cells, such as Tcells are combined with agent-coated or conjugated beads, the beads andthe cells are subsequently separated, and then the cells are cultured.In an alternative embodiment, prior to culture, the agent-coated orconjugated beads and cells are not separated but are cultured together.In a further embodiment, the beads and cells are first concentrated byapplication of a force, resulting in cell surface moiety ligation,thereby inducing cell stimulation and/or polarization of the activationsignal.

By way of example, when T cells are the target cell population, the cellsurface moieties may be ligated by allowing paramagnetic beads to whichanti-CD3 and anti-CD28 antibodies are attached (3×28 beads) to contactthe T cells prepared. In one embodiment the cells (for example, 104 to109 T cells) and beads (for example, DYNABEADS® M-450 CD3/CD28 Tparamagnetic beads at a ratio of 1:1) are combined in a buffer,preferably PBS (without divalent cations such as, calcium andmagnesium). Again, those of ordinary skill in the art can readilyappreciate any cell concentration may be used. For example, the targetcell may be very rare in the sample and comprise only 0.01% of thesample or the entire sample (i.e. 100%) may comprise the target cell ofinterest. Accordingly, any cell number is within the context of thepresent invention. In certain embodiments, it may be desirable tosignificantly decrease the volume in which particles and cells are mixedtogether (i.e., increase the concentration of cells), to ensure maximumcontact of cells and particles. For example, in one embodiment, aconcentration of about 2 billion cells/ml is used. In anotherembodiment, greater than 100 million cells/ml is used. In a furtherembodiment, a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45,or 50 million cells/ml is used. In yet another embodiment, aconcentration of cells from 75, 80, 85, 90, 95, or 100 million cells/mlis used. In further embodiments, concentrations of 125 or 150 millioncells/ml can be used. Using high concentrations can result in increasedcell yield, cell activation, and cell expansion. Further, use of highcell concentrations allows more efficient capture of cells that mayweakly express target antigens of interest, such as CD28-negative Tcells. Such populations of cells may have therapeutic value and would bedesirable to obtain. For example, using high concentration of cellsallows more efficient selection of CD8+ T cells that normally haveweaker CD28 expression.

In a related embodiment, it may be desirable to use lower concentrationsof cells. By significantly diluting the mixture of T cells andparticles, interactions between particles and cells is minimized. Thisselects for cells that express high amounts of desired antigens to bebound to the particles. For example, CD4+ T cells express higher levelsof CD28 and are more efficiently captured and stimulated than CD8+ Tcells in dilute concentrations. In one embodiment, the concentration ofcells used is about 5×10⁶/ml. In other embodiments, the concentrationused can be from about 1×10⁵/ml to about 1×10⁶/ml, and any integer valuein between.

The buffer that the cells are suspended in may be any that isappropriate for the particular cell type. When utilizing certain celltypes the buffer may contain other components, e.g. 1-5% serum,necessary to maintain cell integrity during the process. In anotherembodiment, the cells and beads may be combined in cell culture media.The cells and beads may be mixed, for example, by rotation, agitation orany means for mixing, for a period of time ranging from one minute toseveral hours. The container of beads and cells is then concentrated bya force, such as placing in a magnetic field. Media and unbound cellsare removed and the cells attached to the beads or other surface arewashed, for example, by pumping via a peristaltic pump, and thenresuspended in media appropriate for cell culture.

In one embodiment of the present invention, the mixture may be culturedfor 30 minutes to several hours (about 3 hours) to about 14 days or anyhourly or minute integer value in between. In another embodiment, themixture may be cultured for 21 days. In one embodiment of the inventionthe beads and the T cells are cultured together for about eight days. Inanother embodiment, the beads and T cells are cultured together for 2-3days. As described above, several cycles of stimulation may also bedesired such that culture time of T cells can be 60 days or more.Conditions appropriate for T cell culture include an appropriate media(e.g., Minimal Essential Media or RPMI Media 1640 or, X-vivo 15,(BioWhittaker)) that may contain factors necessary for proliferation andviability, including serum (e.g., fetal bovine or human serum) orinterleukin-2 (IL-2). insulin, or any other additives for the growth ofcells known to the skilled artisan. Media can include RPMI 1640, AIM-V,DMEM, MEM, α-MEM, F-12, X-Vivo 15, and X-Vivo 20, with added amino acidsand vitamins, either serum-free or supplemented with an appropriateamount of serum (or plasma) or a defined set of hormones, and/or anamount of cytokine(s) sufficient for the growth and expansion of Tcells. Antibiotics, e.g., penicillin and streptomycin, are included onlyin experimental cultures, not in cultures of cells that are to beinfused into a subject. The target cells are maintained under conditionsnecessary to support growth, for example, an appropriate temperature(e.g., 37° C.) and atmosphere (e.g., air plus 5% CO₂).

In one embodiment of the present invention, bead:cell ratios can betailored to obtain a desired T cell phenotype. In one particularembodiment, bead:cell ratios can be vaired to selectively expand ordelete antigen-specific (memory) T cells. In one embodiment, theparticular bead:cell ratio used selectively deletes antigen-specific Tcells. In a further embodiment, the particular bead:cell ratio usedselectively expands antigen-specific T cells. The skilled artisan wouldreadily appreciate that any ratio can be used as long as the desiredexpansion or deletion of antigen-specific T cells occurs. Therefore, thecompositions and methods described herein can be used to expand specificpopulations of T cells, or to delete specific populations of T cells,for use in any variety of immunotherapeutic settings described herein.

In another embodiment, the time of exposure to stimulatory agents suchas anti-CD3/anti-CD28 (i.e., 3×28)-coated beads may be modified ortailored in such a way to obtain a desired T cell phenotype.Alternatively, a desired population of T cells can be selected using anynumber of selection techniques, prior to stimulation. One may desire agreater population of helper T cells (T_(H)), typically CD4⁺ as opposedto CD8⁺ cytotoxic or regulatory T cells, because an expansion of T_(H)cells could improve or restore overall immune responsiveness. While manyspecific immune responses are mediated by CD8⁺ antigen-specific T cells,which can directly lyse or kill target cells, most immune responsesrequire the help of CD4⁺ T cells, which express importantimmune-regulatory molecules, such as GM-CSF, CD40L, and IL-2, forexample. Where CD4-mediated help is preferred, a method, such as thatdescribed herein, which preserves or enhances the CD4:CD8 ratio could beof significant benefit. Increased numbers of CD4⁺ T cells can increasethe amount of cell-expressed CD40L introduced into patients, potentiallyimproving target cell visibility (improved APC function). Similareffects can be seen by increasing the number of infused cells expressingGM-CSF, or IL-2, all of which are expressed predominantly by CD4⁺ Tcells. Likewise, it may be desirable in certain applications to utilizea population of regulatory T cells (e.g., Autoimmun Rev. 2002August;1(4):190-7; Curr Opin Immunol. 2002 December;14(6):771-8) whichcan be generated and expanded using the methods described herein.Alternatively, in situations where CD4-help is needed less and increasednumbers of CD8⁺ T cells are desirous, the XCELLERATE™ approachesdescribed herein can also be utilized, by for example, pre-selecting forCD8⁺ cells prior to stimulation and/or culture. Such situations mayexist where increased levels of IFN-γ or increased cytolysis of a targetcell is preferred. One may also modify time and type of exposure tostimulatory agents to expand T cells with a desired TCR repertoire, e.g.expressing desired Vβ family genes.

To effectuate isolation of different T cell populations, exposure timesto the particles may be varied. For example, in one preferredembodiment, T cells are isolated by incubation with 3×28 beads, such asDYNABEADS® M-450, for a time period sufficient for positive selection ofthe desired T cells. In one embodiment, the time period is about 30minutes. In a further embodiment, the time period is at least 1, 2, 3,4, 5, or 6 hours. In yet another preferred embodiment, the time periodis 10 to 24 hours or more. In one preferred embodiment, the incubationtime period is 24 hours. For isolation of T cells from cancer patients,use of longer incubation times, such as 24 hours, can increase cellyield.

In certain embodiments, stimulation and/or expansion times may be 10weeks or less, 8 weeks or less, four weeks or less, 2 weeks or less, 10days or less, or 8 days or less (four weeks or less includes all timeranges from 4 weeks down to 1 day (24 hours) or any value between thesenumbers). In some embodiments in may be desirable to clone T cellsusing, for example, limiting dilution or cell sorting, wherein longerstimulation time may be necessary. In some embodiments, stimulation andexpansion may be carried out for 6 days or less, 4 days or less, 2 daysor less, and in other embodiments for as little as 24 or less hours, andpreferably 4-6 hours or less (these ranges include any integer values inbetween). When stimulation of T cells is carried out for shorter periodsof time, the population of T cells may not increase in number asdramatically, but the population will provide more robust and healthyactivated T cells that can continue to proliferate in vivo and moreclosely resemble the natural effector T cell pool. As the availabilityof T cell help is often the limiting factor in antibody responses toprotein antigens, the ability to selectively expand or selectivelyinfuse a CD4⁺ rich population of T cells into a subject is extremelybeneficial. Further benefits of such enriched populations are readilyapparent in that activated helper T cells that recognize antigenspresented by B lymphocytes deliver two types of stimuli, physicalcontact and cytokine production, that result in the proliferation anddifferentiation of B cells.

In the various embodiments, one of ordinary skill in the art understandsremoval of the stimulation signal from the cells is dependent upon thetype of surface used. For example, if paramagnetic beads are used, thenmagnetic separation is the feasible option. Separation techniques aredescribed in detail by paramagnetic bead manufacturers' instructions(for example, DYNAL Inc., Oslo, Norway). Furthermore, filtration may beused if the surface is a bead large enough to be separated from thecells. In addition, a variety of transfusion filters are commerciallyavailable, including 20 micron and 80 micron transfusion filters(Baxter). Accordingly, so long as the beads are larger than the meshsize of the filter, such filtration is highly efficient. In a relatedembodiment, the beads may pass through the filter, but cells may remain,thus allowing separation. In one particular embodiment, thebiocompatible surface used degrades (i.e. is biodegradable) in cultureduring the exposure period.

Although the antibodies used in the methods described herein can bereadily obtained from public sources, such as the American Type CultureCollection (ATCC), antibodies to T cell accessory molecules and the CD3complex can be produced by standard techniques. Methodologies forgenerating antibodies for use in the methods of the invention arewell-known in the art and are discussed in further detail herein.

Ligand Immobilization on a Surface

As indicated above, the methods of the present invention preferably useligands bound to a surface. The surface may be any surface capable ofhaving a ligand bound thereto or integrated into and that isbiocompatible, that is, substantially non-toxic to the target cells tobe stimulated. The biocompatible surface may be biodegradable ornon-biodegradable. The surface may be natural or synthetic, and asynthetic surface may be a polymer. The surface may comprise collagen,purified proteins, purified peptides, polysaccharides,glycosaminoglycans, extracellular matrix compositions, liposomes, orcell surfaces. A polysaccharide may include for example, cellulose,agarose, dextran, chitosan, hyaluronic acid, or alginate. Other polymersmay include polyesters, polyethers, polyanhydrides,polyalkylcyanoacryllates, polyacrylamides, polyorthoesters,polyphosphazenes, polyvinylacetates, block copolymers, polypropylene,polytetrafluorethylene (PTFE), or polyurethanes. The polymer may belactic acid or a copolymer. A copolymer may comprise lactic acid andglycolic acid (PLGA). Non-biodegradable surfaces may include polymers,such as poly(dimethylsiloxane) and poly(ethylene-vinyl acetate).Biocompatible surfaces include for example, glass (e.g., bioglass),collagen, chitin, metal, hydroxyapatite, aluminate, bioceramicmaterials, hyaluronic acid polymers, alginate, acrylic ester polymers,lactic acid polymer, glycolic acid polymer, lactic acid/glycolic acidpolymer, purified proteins, purified peptides, or extracellular matrixcompositions. Other polymers comprising a surface may include glass,silica, silicon, hydroxyapatite, hydrogels, collagen, acrolein,polyacrylamide, polypropylene, polystyrene, nylon, or any number ofplastics or synthetic organic polymers, or the like. The surface maycomprise a biological structure, such as a liposome or cell surface,such as red blood cells (RBCs). The surface may be in the form of alipid, a plate, bag, pellet, fiber, mesh, or particle. A particle mayinclude, a colloidal particle, a microsphere, nanoparticle, a bead, orthe like. In the various embodiments, commercially available surfaces,such as beads or other particles, are useful (e.g., Miltenyi Particles,Miltenyi Biotec, Germany; Sepharose beads, Pharmacia Fine Chemicals,Sweden; DYNABEADS™, Dynal Inc., New York; PURABEADS™, PrometicBiosciences).

When beads are used, the bead may be of any size that effectuates targetcell stimulation. In one embodiment, beads are preferably from about 5nanometers to about 500 μm in size. Accordingly, the choice of bead sizedepends on the particular use the bead will serve. For example, if thebead is used for monocyte depletion, a small size is chosen tofacilitate monocyte ingestion (e.g., 1.0 μm and 4.5 μm in diameter orany size that may be engulfed, such as nanometer sizes); however, whenseparation of beads by filtration is desired, bead sizes of no less than50 μm are typically used. Further, when using paramagnetic beads, thebeads typically range in size from about 2.8 μm to about 500 μm and morepreferably from about 2.8 μm to about 50 μm. Lastly, one may choose touse super-paramagnetic nanoparticles which can be as small as about 10⁻⁵nm. Accordingly, as is readily apparent from the discussion above,virtually any particle size may be utilized.

An agent may be attached, incorporated into, coupled to, or integratedinto a surface by a variety of methods known and available in the art.The agent may be a natural ligand, a protein ligand, or a syntheticligand. The attachment may be covalent or noncovalent, electrostatic, orhydrophobic and may be accomplished by a variety of attachment means,including for example, chemical, mechanical, enzymatic, electrostatic,or other means whereby a ligand is capable of stimulating the cells. Theattachment of the agent may be direct or indirect (e.g. tethered). Forexample, the antibody to a ligand first may be attached to a surface(direct attachment), or avidin or streptavidin, or a second antibodythat binds the first, may be attached to the surface for binding to abiotinylated ligand (indirect attachment). With respect to cellsurfaces, the attachment may be via genetic expression of the agentusing any number of technologies known in the art, such as transfectionor transduction, etc of an expression vector comprising the codingregion of the agent of interest. The antibody to the ligand may beattached to the surface via an anti-idiotype antibody. Another exampleincludes using protein A or protein G, or other non-specific antibodybinding molecules, attached to surfaces to bind an antibody.Alternatively, the ligand may be attached to the surface by chemicalmeans, such as cross-linking to the surface, using commerciallyavailable cross-linking reagents (Pierce, Rockford, Ill.) or othermeans. In certain embodiments, the ligands are covalently bound to thesurface. Further, in one embodiment, commercially availabletosyl-activated DYNABEADS™ or DYNABEADS™ with epoxy-surface reactivegroups are incubated with the polypeptide ligand of interest accordingto the manufacturer's instructions. Briefly, such conditions typicallyinvolve incubation in a phosphate buffer from pH 4 to pH 9.5 attemperatures ranging from 4 to 37 degrees C.

In one aspect, the agent, such as certain ligands may be of singularorigin or multiple origins and may be antibodies or fragments thereofwhile in another aspect, when utilizing T cells, the co-stimulatoryligand is a B7 molecule (e.g., B7-1, B7-2). These ligands are coupled tothe surface by any of the different attachment means discussed above.The B7 molecule to be coupled to the surface may be isolated from a cellexpressing the co-stimulatory molecule, or obtained using standardrecombinant DNA technology and expression systems that allow forproduction and isolation of the co-stimulatory molecule(s) as describedherein. Fragments, mutants, or variants of a B7 molecule that retain thecapability to trigger a co-stimulatory signal in T cells when coupled tothe surface of a cell can also be used. Furthermore, one of ordinaryskill in the art will recognize that any ligand useful in the activationand induction of proliferation of a subset of T cells may also beimmobilized on beads or culture vessel surfaces or any surface. Inaddition, while covalent binding of the ligand to the surface is onepreferred methodology, adsorption or capture by a secondary monoclonalantibody may also be used. The amount of a particular ligand attached toa surface may be readily determined by flow cytometric analysis if thesurface is that of beads or determined by enzyme-linked immunosorbantassay (ELISA) if the surface is a tissue culture dish, mesh, fibers,bags, for example.

In a particular embodiment, the stimulatory form of a B7 molecule or ananti-CD28 antibody or fragment thereof is attached to the same solidphase surface as the agent that stimulates the TCR/CD3 complex, such asan anti-CD3 antibody. In addition to anti-CD3 antibodies, otherantibodies that bind to receptors that mimic antigen signals may beused. For example, the beads or other surfaces may be coated withcombinations of anti-CD2 antibodies and a B7 molecule and in particularanti-CD3 antibodies and anti-CD28 antibodies.

When coupled to a surface, the agents may be coupled to the same surface(i.e., in “cis” formation) or to separate surfaces (i.e., in “trans”formation). Alternatively, one agent may be coupled to a surface and theother agent in solution. In one embodiment, the agent providing theco-stimulatory signal is bound to a cell surface and the agent providingthe primary activation signal is in solution or coupled to a surface. Ina preferred embodiment, the two agents are immobilized on beads, eitheron the same bead, i.e., “cis,” or to separate beads, i.e., “trans.” Byway of example, the agent providing the primary activation signal is ananti-CD3 antibody and the agent providing the co-stimulatory signal isan anti-CD28 antibody; and both agents are co-immobilized to the samebead in equivalent molecular amounts. In one embodiment, a 1:1 ratio ofeach antibody bound to the beads for CD4⁺ T cell expansion and T cellgrowth is used. In certain aspects of the present invention, a ratio ofanti CD3:CD28 antibodies bound to the beads is used such that anincrease in T cell expansion is observed as compared to the expansionobserved using a ratio of 1:1. In one particular embodiment an increaseof from about 0.5 to about 3 fold is observed as compared to theexpansion observed using a ratio of 1:1. In one embodiment, the ratio ofCD3:CD28 antibody bound to the beads ranges from 100:1 to 1:100 and allinteger values there between. In one aspect of the present invention,more anti-CD28 antibody is bound to the particles than anti-CD3antibody, i.e. the ratio of CD3:CD28 is less than one. In certainembodiments of the invention, the ratio of anti CD28 antibody to antiCD3 antibody bound to the beads is greater than 2:1. In one particularembodiment, a 1:200 CD3:CD28 ratio of antibody bound to beads is used.In one particular embodiment, a 1:150 CD3:CD28 ratio of antibody boundto beads is used. In one particular embodiment, a 1:100 CD3:CD28 ratioof antibody bound to beads is used. In another embodiment, a 1:75CD3:CD28 ratio of antibody bound to beads is used. In a furtherembodiment, a 1:50 CD3:CD28 ratio of antibody bound to beads is used. Inanother embodiment, a 1:45 CD3:CD28 ratio of antibody bound to beads isused. In another embodiment, a 1:40 CD3:CD28 ratio of antibody bound tobeads is used. In another embodiment, a 1:35 CD3:CD28 ratio of antibodybound to beads is used. In another embodiment, a 1:30 CD3:CD28 ratio ofantibody bound to beads is used. In another embodiment, a 1:25 CD3:CD28ratio of antibody bound to beads is used. In another embodiment, a 1:20CD3:CD28 ratio of antibody bound to beads is used. In anotherembodiment, a 1:15 CD3:CD28 ratio of antibody bound to beads is used. Inone preferred embodiment, a 1:10 CD3:CD28 ratio of antibody bound tobeads is used. In another embodiment, a 1:5 CD3:CD28 ratio of antibodybound to beads is used. In another embodiment, a 1:4 CD3:CD28 ratio ofantibody bound to beads is used. In another embodiment, a 1:3 CD3:CD28ratio of antibody bound to the beads is used. In yet another embodiment,a 3:1 CD3:CD28 ratio of antibody bound to the beads is used.

Surface-Associated Agents

Agents contemplated by the present invention include protein ligands,natural ligands, and synthetic ligands. Agents that can bind to cellsurface moieties, and under certain conditions, cause ligation andaggregation that leads to signaling include, but are not limited to,lectins (for example, phyotohaemagluttinin (PHA), lentil lectins,concanavalin A), antibodies, antibody fragments, peptides, polypeptides,glycopeptides, receptors, B cell receptor and T cell receptor ligands,MHC-peptide dimers or tetramers, extracellular matrix components,steroids, hormones (for example, growth hormone, corticosteroids,prostaglandins, tetra-iodo thyronine), bacterial moieties (such aslipopolysaccharides), mitogens, superantigens and their derivatives,growth factors, cytokines, adhesion molecules (such as, L-selectin,LFA-3, CD54, LFA-1), chemokines, and small molecules. The agents may beisolated from natural sources such as cells, blood products, andtissues, or isolated from cells propogated in vitro, preparedrecombinantly, by chemical synthesis, or by other methods known to thosewith skill in the art.

In one aspect of the present invention, when it is desirous to stimulateT cells, useful agents include ligands that are capable of binding theCD3/TCR complex, CD2, and/or CD28 and initiating activation orproliferation, respectively. Accordingly, the term ligand includes thoseproteins that are the “natural” ligand for the cell surface protein,such as a B7 molecule for CD28, as well as artificial ligands such asantibodies directed to the cell surface protein. Such antibodies andfragments thereof may be produced in accordance with conventionaltechniques, such as hybridoma methods and recombinant DNA and proteinexpression techniques. Useful antibodies and fragments may be derivedfrom any species, including humans, or may be formed as chimericproteins, which employ sequences from more than one species.

Methods well known in the art may be used to generate antibodies,polyclonal antisera, or monoclonal antibodies that are specific for aligand. Antibodies also may be produced as genetically engineeredimmunoglobulins (Ig) or Ig fragments designed to have desirableproperties. For example, by way of illustration and not limitation,antibodies may include a recombinant IgG that is a chimeric fusionprotein having at least one variable (V) region domain from a firstmammalian species and at least one constant region domain from a seconddistinct mammalian species. Most commonly, a chimeric antibody hasmurine variable region sequences and human constant region sequences.Such a murine/human chimeric immunoglobulin may be “humanized” bygrafting the complementarity determining regions (CDRs), which conferbinding specificity for an antigen, derived from a murine antibody intohuman-derived V region framework regions and human-derived constantregions. Antibodies containing CDRs of different specificities can alsobe combined to generate multi-specific (bi or tri-specific, etc.)antibodies. Fragments of these molecules may be generated by proteolyticdigestion, or optionally, by proteolytic digestion followed by mildreduction of disulfide bonds and alkylation, or by recombinant geneticengineering techniques.

Antibodies are defined to be “immunospecific” if they specifically bindthe antigen with an affinity constant, K_(a), of greater than or equalto about 10⁴ M⁻¹, preferably of greater than or equal to about 10⁵ M⁻¹,more preferably of greater than or equal to about 10⁶ M⁻¹, and stillmore preferably of greater than or equal to about 10⁷ M⁻¹. Affinities ofbinding partners or antibodies can be readily determined usingconventional techniques, for example, those described by Scatchard etal. (Ann. N.Y. Acad. Sci. USA 51:660, 1949) or by surface plasmonresonance (BIAcore, Biosensor, Piscataway, N.J.) See, e.g., Wolff etal., Cancer Res., 53:2560-2565, 1993).

Antibodies may generally be prepared by any of a variety of techniquesknown to those having ordinary skill in the art (See, e.g., Harlow etal., Antibodies: A Laboratory Manual, 1988, Cold Spring HarborLaboratory). In one such technique, an animal is immunized with theligand as antigen to generate polyclonal antisera. Suitable animalsinclude rabbits, sheep, goats, pigs, cattle, and may include smallermammalian species, such as, mice, rats, and hamsters. Antibodies of thepresent invention may also be generated as described in U.S. Pat. Nos.6,150,584, 6,130,364, 6,114,598, 5,833,985, 6,071,517, 5,756,096,5,736,137, and 5,837,243.

An immunogen may be comprised of cells expressing the ligand, purifiedor partially purified ligand polypeptides or variants or fragmentsthereof, or ligand peptides. Ligand peptides may be generated byproteolytic cleavage or may be chemically synthesized. Peptides forimmunization may be selected by analyzing the primary, secondary, ortertiary structure of the ligand according to methods know to thoseskilled in the art in order to determine amino acid sequences morelikely to generate an antigenic response in a host animal (See, e.g.,Novotny, Mol. Immunol. 28:201-207, 1991; Berzoksky, Science 229:932-40,1985).

Preparation of the immunogen may include covalent coupling of the ligandpolypeptide or variant or fragment thereof, or peptide to anotherimmunogenic protein, such as, keyhole limpet hemocyanin or bovine serumalbumin. In addition, the peptide, polypeptide, or cells may beemulsified in an adjuvant (See Harlow et al., Antibodies: A LaboratoryManual, 1988 Cold Spring Harbor Laboratory). In general, after the firstinjection, animals receive one or more booster immunizations accordingto a preferable schedule for the animal species. The immune response maybe monitored by periodically bleeding the animal, separating the sera,and analyzing the sera in an immunoassay, such as an Ouchterlony assay,to assess the specific antibody titer. Once an antibody titer isestablished, the animals may be bled periodically to accumulate thepolyclonal antisera. Polyclonal antibodies that bind specifically to theligand polypeptide or peptide may then be purified from such antisera,for example, by affinity chromatography using protein A or using theligand polypeptide or peptide coupled to a suitable solid support.

Monoclonal antibodies that specifically bind ligand polypeptides orfragments or variants thereof may be prepared, for example, using thetechnique of Kohler and Milstein (Nature, 256:495-497, 1975; Eur. J.Immunol. 6:511-519, 1976) and improvements thereto. Hybridomas, whichare immortal eucaryotic cell lines, may be generated that produceantibodies having the desired specificity to a ligand polypeptide orvariant or fragment thereof. An animal—for example, a rat, hamster, orpreferably mouse—is immunized with the ligand immunogen prepared asdescribed above. Lymphoid cells, most commonly, spleen cells, obtainedfrom an immunized animal may be immortalized by fusion with adrug-sensitized myeloma cell fusion partner, preferably one that issyngeneic with the immunized animal. The spleen cells and myeloma cellsmay be combined for a few minutes with a membrane fusion-promotingagent, such as polyethylene glycol or a nonionic detergent, and thenplated at low density on a selective medium that supports the growth ofhybridoma cells, but not myeloma cells. A preferred selection media isHAT (hypoxanthine, aminopterin, thymidine). After a sufficient time,usually about 1 to 2 weeks, colonies of cells are observed. Singlecolonies are isolated, and antibodies produced by the cells may betested for binding activity to the ligand polypeptide or variant orfragment thereof. Hybridomas producing antibody with high affinity andspecificity for the ligand antigen are preferred. Hybridomas thatproduce monoclonal antibodies that specifically bind to a ligandpolypeptide or variant or fragment thereof are contemplated by thepresent invention.

Monoclonal antibodies may be isolated from the supernatants of hybridomacultures. An alternative method for production of a murine monoclonalantibody is to inject the hybridoma cells into the peritoneal cavity ofa syngeneic mouse. The mouse produces ascites fluid containing themonoclonal antibody. Contaminants may be removed from the antibody byconventional techniques, such as chromatography, gel filtration,precipitation, or extraction.

Human monoclonal antibodies may be generated by any number oftechniques. Methods include but are not limited to, Epstein Barr Virus(EBV) transformation of human peripheral blood cells (see, U.S. Pat. No.4,464,456), in vitro immunization of human B cells (see, e.g., Boerneret al., J. Immunol. 147:86-95, 1991), fusion of spleen cells fromimmunized transgenic mice carrying human immunoglobulin genes and fusionof spleen cells from immunized transgenic mice carrying immunoglobulingenes inserted by yeast artificial chromosome (YAC) (see, e.g., U.S.Pat. No. 5,877,397; Bruggemann et al., Curr. Opin. Biotechnol. 8:455-58,1997; Jakobovits et al., Ann. N.Y. Acad. Sci. 764:525-35, 1995), orisolation from human immunoglobulin V region phage libraries.

Chimeric antibodies and humanized antibodies for use in the presentinvention may be generated. A chimeric antibody has at least oneconstant region domain derived from a first mammalian species and atleast one variable region domain derived from a second distinctmammalian species (See, e.g., Morrison et al., Proc. Natl. Acad. Sci.USA, 81:6851-55, 1984). Most commonly, a chimeric antibody may beconstructed by cloning the polynucleotide sequences that encode at leastone variable region domain derived from a non-human monoclonal antibody,such as the variable region derived from a murine, rat, or hamstermonoclonal antibody, into a vector containing sequences that encode atleast one human constant region. (See, e.g., Shin et al., MethodsEnzymol. 178:459-76, 1989; Walls et al., Nucleic Acids Res. 21:2921-29,1993). The human constant region chosen may depend upon the effectorfunctions desired for the particular antibody. Another method known inthe art for generating chimeric antibodies is homologous recombination(U.S. Pat. No. 5,482,856). Preferably, the vectors will be transfectedinto eukaryotic cells for stable expression of the chimeric antibody.

A non-human/human chimeric antibody may be further geneticallyengineered to create a “humanized” antibody. Such an antibody has aplurality of CDRs derived from an immunoglobulin of a non-humanmammalian species, at least one human variable framework region, and atleast one human immunoglobulin constant region. Humanization may yieldan antibody that has decreased binding affinity when compared with thenon-human monoclonal antibody or the chimeric antibody. Those havingskill in the art, therefore, use one or more strategies to designhumanized antibodies.

Within certain embodiments, the use of antigen-binding fragments ofantibodies may be preferred. Such fragments include Fab fragments orF(ab′)₂ fragments, which may be prepared by proteolytic digestion withpapain or pepsin, respectively. The antigen binding fragments may beseparated from the Fc fragments by affinity chromatography, for example,using immobilized protein A or immobilized ligand polypeptide or avariant or a fragment thereof. An alternative method to generate Fabfragments includes mild reduction of F(ab′)₂ fragments followed byalkylation (See, e.g., Weir, Handbook of Experimental Immunology, 1986,Blackwell Scientific, Boston).

Non-human, human, or humanized heavy chain and light chain variableregions of any of the above described Ig molecules may be constructed assingle chain Fv (sFv) fragments (single chain antibodies). See, e.g.,Bird et al., Science 242:423-426, 1988; Huston et al., Proc. Natl. Acad.Sci. USA 85:5879-5883, 1988. Multi-functional fusion proteins may begenerated by linking polynucleotide sequences encoding an sFv in-framewith polynucleotide sequences encoding various effector proteins. Thesemethods are known in the art, and are disclosed, for example, inEP-B1-0318554, U.S. Pat. No. 5,132,405, U.S. Pat. No. 5,091,513, andU.S. Pat. No. 5,476,786.

An additional method for selecting antibodies that specifically bind toa ligand polypeptide or variant or fragment thereof is by phage display(See, e.g., Winter et al., Annul. Rev. Immunol. 12:433-55, 1994; Burtonet al., Adv. Immunol. 57:191-280, 1994). Human or murine immunoglobulinvariable region gene combinatorial libraries may be created in phagevectors that can be screened to select Ig fragments (Fab, Fv, sFv, ormultimers thereof) that bind specifically to a ligand polypeptide orvariant or fragment thereof (See, e.g., U.S. Pat. No. 5,223,409; Huse etal., Science 246:1275-81, 1989; Kang et al., Proc. Natl. Acad. Sci. USA88:4363-66, 1991; Hoogenboom et al., J. Molec. Biol. 227:381-388, 1992;Schlebusch et al., Hybridoma 16:47-52, 1997 and references citedtherein).

Methods of Use

Monoclonal and oligoclonal T cell populations are associated with mostautoimmune diseases and are often correlated with disease activity.Further, broad T cell repertoire is restored when patients achievedisease remission. The present invention relates generally to methodsfor stimulating T cells, and more particularly, to methods to eliminateundesired (e.g. autoreactive, alloreactive, pathogenic) subpopulationsof T cells from a mixed population of T cells, thereby restoring thenormal immune repertoire of the T cells. Thus, the present inventionprovides compositions of cells, including stimulated T cells havingrestored immune repertoire and uses thereof.

Generally, the compositions and methodologies described herein can beused to to eliminate at least a portion of undesired clonal populationsof cells, typically T cells, B cells, NKT, or NK cells, from apopulation of immune cells. The present invention further provides forcompositions comprising populations of cells that no longer containundesired cells, or have a significantly reduced number of undesiredcells, and uses thereof. The compositions and methods of the presentinvention are also used to selectively expand a population of cells thathave been deleted for undesired clonal populations for use in thetreatment of immune defects associated with hematopoietic stem celltransplantation (including allotransplantation and autotransplantationfrom sources that include blood, cord blood, and bone marrow), organtransplantation (e.g., acute or chronic GVHD), and autoimmune diseases,including autoimmune disease caused by cancers such as large granularlymphocyte (LGL) leukemia, chronic lymphocytic leukemia (CLL) or bycommon variable immunodeficiency. As a result, a population of cells, inthe case of T cells, that express TCRs that are polyclonal with respectto antigen reactivity, but essentially homogeneous with respect toeither CD4⁺ or CD8⁺, can be produced that have been cleared of anyundesired subpopulations of cells, such as autoreactive cells oralloreactive cells. With respect to B cells, a populations of cells canbe produced that has been cleared of any undesired subpopulations of Bcells producing autoreactive antibodies. In addition, the method allowsfor the expansion of the resulting population of T- or B-cells innumbers sufficient to reconstitute an individual's total CD4⁺ or CD8⁺ Tcell population or B cell population (the population of lymphocytes inan individual is approximately 5×10¹¹ cells). The resulting cellpopulation can also be genetically transduced using a variety oftechniques known to the skilled artisan and used for immunotherapy.

In one embodiment, the T or B cell compositions of the present inventionmay be used in the context of hematopoietic stem cell transplantation.The major problem in hematopoietic stem cell transplantation isgraft-versus-host disease (GVHD), which is caused by alloreactive Tcells present in the infused hematopoietic stem cell preparation. Thus,the present invention may be used to remove alloreative T cells and toexpand the remaining T cell population for infusion into the patient.The cell compositions of the present invention can be used alone or inconjunction with other therapies.

In one embodiment, the T or B cell compositions of the present inventionmay be used in the context of any autoimmune disease. Illustrativeautoimmune diseases include, but are not limited to, systemic lupuserythematosus (SLE), multiple sclerosis (MS), rheumatoid arthritis,progressive systemic sclerosis, Sjogren's syndrome, multiple sclerosis,polymyositis, dermatomyositis, uveitis, arthritis, psoriatic arthritis,reactive arthritis, Type I insulin-dependent diabetes, Hashimoto'sthyroiditis, Grave's thyroiditis, myasthenia gravis, autoimmunemyocarditis, vasculitis, aplastic anemia, autoimmune hemolytic anemia,myelodysplastic syndrome, Evan's syndrome, stiff person syndrome, atopicdermatitis, psoriasis, Behchet's syndrome, Crohn's disease, biliarycirrhosis, inflammatory bowel disease, ulcerative colitis, Goodpasture'ssyndrome, Wegener's granulomatosis, paroxysmal nocturnal hemaglobinuria,myelodysplastic syndrome, allergic disorders such as hay fever,extrinsic asthma, or insect bite and sting allergies, and food and drugallergies.

Further uses of the T and B cell compositions of the present inventionmay include the treatment and/or prophylaxis of: inflammatory andhyperproliferative skin diseases and cutaneous manifestations ofimmunologically mediated illnesses, such as, seborrhoeis dermatitis,angioedemas, erythemas, acne, and Alopecia greata; various eye diseases(autoimmune and otherwise); allergic reactions, such as pollenallergies, reversible obstructive airway disease, which includescondition such as asthma (for example, bronchial asthma, allergicasthma, intrinsic asthma, extrinsic asthma and dust asthma),particularly chronic or inveterate asthma (for example, late asthma andairway hyper-responsiveness), bronchitis, allergic rhinitis, and thelike; inflammation of mucous and blood vessels.

As noted above, the T and B cell compositions of the present inventionmay be used in the treatment of immune defects associated with organtransplantation, e.g., host versus graft disease. Treatment of immunedefects associated with any organ transplantation is contemplatedherein. For example, the methods and cells of the present invention canbe used in the treatment of immune defects associated with kidney,heart, lung, and liver transplantation.

In certain embodiments of the present invention, the cells of thepresent invention are administered to a patient following treatment withan agent such as chemotherapy, radiation, immunosuppressive agents, suchas cyclosporine, azathioprine, methotrexate, mycophenolate, and FK506,antibodies, or other immunoablative agents such as CAMPATH, anti-CD3antibodies, cyclophosphamide, fludarabine, cyclosporine, FK506,rapamycin, mycophenolic acid, steroids, FR901228, and irradiation. Thesedrugs inhibit either the calcium dependent phosphatase calcineurin(cyclosporine and FK506) or inhibit the p70S6 kinase that is importantfor growth factor induced signaling (rapamycin). (Liu et al., Cell66:807-815, 1991; Henderson et al., Immun. 73:316-321, 1991; Bierer etal., Curr. Opin. Immun. 5:763-773, 1993; Isoniemi (supra)).

In a further embodiment, the cells are administered to a patient whoseimmune system has been rendered essentially to a naive state throughtreatment with one or more agents as described herein, followed by anorgan transplant in conjunction with an immunosuppressive regimen.Immunosuppresive regimens useful in this context include but are notlimited to, anti-CD3 antibodies, anti-CD25 antibodies, ATG,thymoglobulin, campath, fludarabine, cyclophosphamide, FK506,mycophenolate, cyclosporine, CTLA-4 IG, anti-CD40 antibody, destruxin,radiation therapy, and the like. In this regard, many immunosuppressiveregimens have been shown to be effective in animal transplant models(e.g., mouse models) but have not been successful in the clinic. Withoutbeing bound by theory, one of the major reasons for this decrepancy isthought to be the presence of antigen-specific memory T cells that crossreact with donor alloantigens in the transplanted organ. Lymphoablationin vivo followed by infusion of activated T cells grown under conditionsthat favor deletion of antigen-specific memory T cells as describedherein while preserving naïve T cells may provide conditions similar tothe pathogen-free mice that accept organs under a variety ofimmunosuppressive regimens. This would enable wider and more successfuluse of immunosuppressive drugs. Additionally, infusion of the T cells asdescribed herein in this setting would lead to a decrease in the use andamount of toxic immunosuppresive drugs, including the number of drugsadministered and the length of time that patients are on these drugs.This in turn would lead to increased efficacy (fewer organ rejections)and decreased toxicity. Further, this would also allow the use of organtransplantation in settings such as severe mismatching and could allowsuccessful transplants in xenogeneic settings.

In a further embodiment, the cell compositions of the present inventionare administered to a patient with autoimmune disease following T cellablative therapy using either chemotherapy agents such as, fludarabine,external-beam radiation therapy (XRT), cyclophosphamide, or antibodiessuch as OKT3 or CAMPATH. In another embodiment, the cell compositions ofthe present invention are administered to a patient with autoimmunedisease following B-cell ablative therapy such as agents that react withCD20, e.g. Rituxan. The dosage of the above treatments to beadministered to a patient will vary with the precise nature of thecondition being treated and the recipient of the treatment. The scalingof dosages for human administration can be performed according toart-accepted practices. The dose for CAMPATH, for example, willgenerally be in the range 1 to about 100 mg for an adult patient,usually administered daily for a period between 1 and 30 days. Thepreferred daily dose is 1 to 10 mg per day although in some instanceslarger doses of up to 40 mg per day may be used (described in U.S. Pat.No. 6,120,766).

In a further aspect of the present invention, at least a substantialportion of autoreactive cells from a patient are eliminated in vitrousing the methods of the present invention then further stimulated andexpanded and administered to the patient. In a related embodiment, atleast a substantial portion of autoreactive cells from a patient areeliminated in vitro using the methods of the present invention thenadministered to the patient and expanded in vivo. It is envisioned asone aspect that the compositions of the present invention can be used inconjunction with other therapies available in the art for treatment ofautoimmune disease.

In one embodiment, T cells can be stimulated and expanded as describedherein to induce or enhance responsiveness in an individual who isimmunocompromised as a result of treatment associated with hematopoieticstem cell transplantation. The present invention provides methods forreducing the risk of, or the severity of, an adverse GVHD effect in apatient who is undergoing a hematopoietic stem cell transplant,comprising administering to the patient a population of T cells of thepresent invention. In one particular embodiment, at least a substantialportion of alloreactive cells present in the donor hematopoietic stemcells are eliminated by the methods of the present invention. In afurther embodiment, the T cell compositions of the present invention areadministered to a patient undergoing a hematopoietic stem celltransplantation following treatment with chemotherapy agents. In afurther embodiment, at least a substantial portion of alloreactive cellsfrom the donor marrow are eliminated in vitro using the methods of thepresent invention then further stimulated and expanded and thenadministered to the patient. In a further embodiment, at least asubstantial portion of alloreactive cells from the donor marrow areeliminated in vitro using the methods of the present invention thenadministered to the patient and expanded in vivo. It is envisioned asone aspect that the compositions of the present invention can be used inconjunction with other therapies available in the art for use inhematopoietic stem cell transplantation, such as administration ofG-CSF, IL-2, IL-11, IL-7, IL-12, and antiviral treatments.

In one embodiment, T cells can be stimulated and expanded as describedherein to induce or enhance responsiveness in an individual who isimmunocompromised as a result of treatment associated with organtransplantation, including but not limited to, kidney, heart, lung, andliver transplantation. In one particular embodiment, at least asubstantial portion of alloreactive cells present in the recipient areeliminated by the methods of the present invention. Thus, the presentinvention provides methods for reducing the risk of, or the severity of,organ rejection. In a further embodiment, the T cell compositions of thepresent invention are administered to a patient undergoing an organtransplant following treatment with chemotherapy agents. In a furtherembodiment, at least a substantial portion of alloreactive cells fromthe transplant recipient are eliminated in vitro using the methods ofthe present invention then further stimulated and expanded and thenadministered to the patient. It is envisioned as one aspect that thecompositions of the present invention can be used in conjunction withother therapies available known in the art for use in organtransplantation.

Another embodiment of the invention, provides a method for selectivelyexpanding a population of T_(H1) cells from a population of CD4⁺ Tcells. In this method, CD4⁺ T cells are co-stimulated with an anti-CD28antibody, such as the monoclonal antibody 9.3, inducing secretion ofT_(H1)-specific cytokines, including IFN-γ, resulting in enrichment ofT_(H1) cells over T_(H2) cells. As described further herein,XCELLERATED™ T cells from patients with autoimmune disease demonstrate aT_(H)1-type phenotype (see FIG. 9). Accordingly, the XCELLERATE™ processcan be used to expand T cells of a desired phenotype, including TH1-typephenotype.

The present invention further provides a method for selectivelyexpanding a population of T_(H2) cells from a population of CD4⁺ Tcells. In this method, CD4⁺ T cells are co-stimulated with an anti-CD28antibody, such as the monoclonal antibody B-T3, XR-CD28, inducingsecretion of T_(H2)-specific cytokines, resulting in enrichment ofT_(H2) cells over T_(H1) cells (see for example, Fowler, et al. Blood1994 Nov. 15;84(10):3540-9; Cohen, et al., Ciba Found Symp1994;187:179-93).

The present invention further provides methods for using the instantcell compositions in conjunction with regulatory T cells. Without beingbound by theory, regulatory T cells may provide help in suppressingaberrant immune responses or otherwise regulating immune cells of thepresent invention. Regulatory T cells can be generated and expandedusing the methods as described herein. The regulatory T cells can beantigen-specific and/or polyclonal. Regulatory T cells can also begenerated using art-recognized techniques as described for example, inWoo, et al., J. Immunol. 2002 May 1;168(9):4272-6; Shevach, E. M., Annu.Rev. Immunol. 2000, 18:423; Stephens, et al., Eur. J. Immunol. 2001,31:1247; Salomon, et al, Immunity 2000, 12:431; and Sakaguchi, et al.,Immunol. Rev. 2001, 182:18.

The present invention further provides a method for selectivelyexpanding a population of T cells expressing a specific Vβ, Vα, Vγ, orVδ gene. For example, in this method, T cells expressing a particularVβ, Vα, Vγ, or Vδ gene are positively or negatively selected and thenfurther expanded/stimulated according to the methods of the presentinvention. Alternatively, stimulated and expanded T cells expressing aparticular Vβ, Vα, Vγ, or Vδ gene of interest can be positively ornegatively selected and further stimulated and expanded.

One aspect of the present invention is to administer activated andexpanded T cells that proliferate and grow rapidly in vivo. Withoutbeing bound by theory, the infused T cells may suppress in vivohomeostatic T cell proliferation and prevent unwanted T cells fromproliferating in vivo, for example cancer cells, autoreactive T cells,alloreactive T cells, HIV infected T cells, and the like (see King, etal., 2004, Cell 117:265-277). Accordingly, the compositions describedherein comprising T cells that have been cultured so as to delete atleast a substantial portion of unwanted cells can be infused into apatient. In one embodiment, the cells are infused at a dose such thatthe cells then prevent homeostatic proliferation and therefore, preventunwanted cells from regenerating in vivo. The frequency ofadministration of the cells of the present invention will be determinedby such factors as the condition of the patient, and the type andseverity of the patient's disease, although appropriate dosages may bedetermined by clinical trials. The precise amount of the compositionscomprising cells of the present invention to be administered can bedetermined by a physician with consideration of individual differencesin age, weight, disease severity and condition of the patient and anyother factors relevant to treatment of the patient.

In another example, blood is drawn into a stand-alone disposable devicedirectly from the patient that contains a sensitizing composition and ortwo or more immobilized antibodies (e.g., anti-CD3 and anti-CD28) orother components to stimulate receptors required for T cell activationprior to the cells being administered to the subject (e.g., immobilizedon plastic surfaces or upon separable microparticles). In oneembodiment, the disposable device may comprise a container (e.g., aplastic bag, or flask) with appropriate tubing connections suitable forcombining/docking with syringes and sterile docking devices. This devicewill contain a solid surface for immobilization of T cell activationcomponents (e.g., anti-CD3 and anti-CD28 antibodies); these may be thesurfaces of the container itself or an insert and will typically be aflat surface, an etched flat surface, an irregular surface, a porouspad, fiber, clinically acceptable/safe ferro-fluid, beads, etc.).Additionally when using the stand-alone device, the subject can remainconnected to the device, or the device can be separable from thepatient. Further, the device may be utilized at room temperature orincubated at physiologic temperature using a portable incubator.

As devices and methods for collecting and processing blood and bloodproducts are well known, one of skill in the art would readily recognizethat given the teachings provided herein, that a variety of devices thatfulfill the needs set forth above may be readily designed or existingdevices modified. Accordingly, as such devices and methods are notlimited by the specific embodiments set forth herein, but would includeany device or methodology capable of maintaining sterility and whichmaintains blood in a fluid form in which complement activation isreduced and wherein components necessary for T cell activation (e.g.,anti-CD3 and anti-CD28 antibodies or ligands thereto) may be immobilizedor separated from the blood or blood product prior to administration tothe subject. Further, as those of ordinary skill in the art can readilyappreciate a variety of blood products can be utilized in conjunctionwith the devices and methods described herein. For example, the methodsand devices could be used to provide rapid activation of T cells fromcryopreserved whole blood, peripheral blood mononuclear cells, othercyropreserved blood-derived cells, or cryopreserved T cell lines uponthaw and prior to subject administration. In another example, themethods and devices can be used to boost the activity of a previously exvivo expanded T cell product or T cell line prior to administration tothe subject, thus providing a highly activated T cell product. Lastly,as will be readily appreciated the methods and devices above may beutilized for autologous or allogeneic cell therapy simultaneously withthe subject and donor.

The methods of the present invention may also be utilized with vaccinesto enhance reactivity of the antigen and enhance in vivo effect. In oneembodiment, the compositions of the present invention are administeredto a patient in conjunction with a composition that enhances T cells invivo, for example, IL-2, IL-4, IL-7, IL-10, IL-12, and IL-15. Further,given that T cells expanded by the present invention have a relativelylong half-life in the body, these cells could act as perfect vehiclesfor gene therapy, by carrying a desired nucleic acid sequence ofinterest and potentially homing to sites of cancer, disease, orinfection. Accordingly, the cells expanded by the present invention maybe delivered to a patient in combination with a vaccine, one or morecytokines, one or more therapeutic antibodies, etc. Virtually anytherapy that would benefit by a more robust T cell population is withinthe context of the methods of use described herein.

A variety of in vitro and animal models exist for testing and validatingthe cell compositions of the present invention and their applicabilityto a particular immune system related disease or indication.Accordingly, one of ordinary skill in the art could easily choose theappropriate model from those currently existing in the art. Such modelsinclude the use of NOD mice, where IDDM results from a spontaneous Tcell dependent autoimmune destruction of insulin-producing pancreatic βcells that intensifies with age (Bottazzo et al., J. Engl. J. Med.,113:353, 1985; Miyazaki et al., Clin. Exp. Immunol., 60:622, 1985). InNOD mice, a model of human IDDM, therapeutic strategies that target Tcells have been successful in preventing IDDM (Makino et al., Exp.Anim., 29:1, 1980). These include neonatal thymectomy, administration ofcyclosporine, and infusion of anti-pan T cell, anti-CD4, or anti-CD25(IL-2R) monoclonal antibodies (mAbs) (Tarui et al., Insulitis and Type IDiabetes Lessons from the NOD Mouse, Academic Press, Tokyo, p.143,1986). Other models include, for example, those typically utilized forautoimmune and inflammatory disease, such as multiple sclerosis (EAEmodel), rheumatoid arthritis, graft-versus-host disease (transplantationmodels for studying graft rejection using skin graft, heart transplant,islet of Langerhans transplants, large and small intestine transplants,and the like), asthma models, systemic lupus erythematosus (systemicautoimmunity—lpr or NZBx NZWF₁ models), and the like. (see, for example,Takakura et al., Exp. Hematol. 27(12):1815-821, 1999; Hu et al.,Immunology 98(3):379-385, 1999; Blyth et al., Am. J. Respir. Cell Mol.Biol. 14(5):425-438, 1996; Theofilopoulos and Dixon, Adv. Immunol.37:269-389, 1985; Eisenberg et al., J. Immunol. 125:1032-1036, 1980;Bonneville et al., Nature 344:163-165, 1990; Dent et al., Nature343:714-719, 1990; Todd et al., Nature 351:542-547, 1991; Watanabe etal., Biochem Genet. 29:325-335, 1991; Morris et al., Clin. Immunol.Immunopathol. 57:263-273, 1990; Takahashi et al., Cell 76:969-976, 1994;Current Protocols in Immunology, Richard Coico (Ed.), John Wiley & Sons,Inc., Chapter 15, 1998).

Collagen-induced arthritis (CIA) is a T cell dependent animal model ofrheumatoid arthritis (RA) (D. E. Trentham et al., “Autoimmunity to TypeII Collagen: An Experimental Model of Arthritis,” J. Exp. Med. 146:857-868 (1977)). Within two weeks after immunization with type IIcollagen (CII) in IFA, susceptible rats develop polyarthritis withhistologic changes of pannus formation and bone/cartilage erosion. Inaddition, humoral and cellular responses to CII occur in CIA as well asRA (E. Brahn, “Animal Models of Rheumatoid Arthritis: Clues to Etiologyand Treatment” in Clinical Orthopedics and Related Research (B. Hahn,ed., Philadelphia, JB Lippincott Company, 1991). Consequently, CIA is auseful animal model of RA that serves as an in vivo system for theinvestigation of potentially new therapeutic interventions as describedin the present invention.

Pharmaceutical Compositions

T cell populations of the present invention may be administered eitheralone, or as a pharmaceutical composition in combination with diluentsand/or with other components such as IL-2 or other cytokines or cellpopulations. Briefly, pharmaceutical compositions of the presentinvention may comprise a target cell population as described herein, incombination with one or more pharmaceutically or physiologicallyacceptable carriers, diluents or excipients. Such compositions maycomprise buffers such as neutral buffered saline, phosphate bufferedsaline and the like; carbohydrates such as glucose, mannose, sucrose ordextrans, mannitol; proteins; polypeptides or amino acids such asglycine; antioxidants; chelating agents such as EDTA or glutathione;adjuvants (e.g., aluminum hydroxide); and preservatives. Compositions ofthe present invention are preferably formulated for intravenousadministration. The present invention further provides forpharmaceutical compositions comprising sensitizing compositions asdescribed herein.

Pharmaceutical compositions of the present invention may be administeredin a manner appropriate to the disease to be treated (or prevented). Thequantity and frequency of administration will be determined by suchfactors as the condition of the patient, and the type and severity ofthe patient's disease, although appropriate dosages may be determined byclinical trials. When “an immunologically effective amount” or“therapeutic amount” is indicated, the precise amount of thecompositions of the present invention to be administered can bedetermined by a physician with consideration of individual differencesin age, weight, disease severity and condition of the patient and anyother factors relevant to treatment of the patient. It can generally bestated that a pharmaceutical composition comprising the subject T or Bcells, may be administered at a dosage of 10⁴ to 10⁷ APC/kg body weight,preferably 10⁵ to 10⁶ APC/kg body weight, including all integer valueswithin those ranges. Cell compositions may also be administered multipletimes at these dosages. The cells can be administered by using infusiontechniques that are commonly known in immunotherapy (see, e.g.,Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimaldosage and treatment regime for a particular patient can readily bedetermined by one skilled in the art of medicine by monitoring thepatient for signs of disease and adjusting the treatment accordingly.

In certain adoptive immunotherapy studies, T cells are administeredapproximately at 1×10⁹ to 2×10¹¹ cells to the patient. (See, e.g., U.S.Pat. No. 5,057,423). In some aspects of the present invention,particularly in the use of allogeneic or xenogeneic cells, lower numbersof cells, in the range of 10⁶/kilogram (10⁶-10¹¹ per patient) may beadministered. In certain embodiments, T or B cells are administered at1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸, 5×10⁸, 1×10⁹, 5×10⁹, 1×10¹⁰, 5×10¹⁰, 1×10¹¹,5×10¹¹, or 1×10¹² cells to the subject. T or B cell compositions may beadministered multiple times at dosages within these ranges. The T or Bcells may be autologous or heterologous (allogeneic or xenogeneic) tothe patient undergoing therapy. If desired, the treatment may alsoinclude administration of mitogens (e.g., PHA) or lymphokines,cytokines, and/or chemokines (e.g., GM-CSF, IL-4, IL-13, Flt3-L, RANTES,MIP1α, etc.) as described herein to enhance restoration of the immuneresponse.

The present invention also provides methods for preventing, inhibiting,or reducing the severity of autoimmune disease in an animal, whichcomprise administering to an animal an effective amount of the subjectactivated polyclonal T cells that have been cleared of undesiredsubpopulations of autoreactive T cells. The T cell compositions of thepresent invention can be administered in conjunction with T cellablative therapy and/or other therapies for the treatment of autoimmunediseases.

The present invention also provides methods for preventing, inhibiting,or reducing the severity of graft-versus-host disease in an animalrequiring a hematopoietic stem cell transplant, which compriseadministering to an animal an effective amount of the subject donor bonemarrow that has been cleared of undesired subpopulations of alloreactiveT cells. The compositions of the present invention can be administeredin conjunction with other therapies for the treatment of immune defectsassociated with hematopoietic stem cell transplantation.

The present invention also provides methods for preventing, inhibiting,or reducing the severity of host-versus-graft disease or graft rejectionin an animal requiring an organ transplant, which comprise administeringto an animal an effective amount of the subject T cell compositions thathas been cleared of undesired subpopulations of alloreactive T cells.The compositions of the present invention can be administered inconjunction with other therapies for the treatment of immune defectsassociated with organ transplantation.

One aspect of the present invention is to administer activated andexpanded T cells that proliferate and grow rapidly in vivo. Withoutbeing bound by theory, the infused T cells may suppress in vivohomeostatic T cell proliferation and prevent unwanted T cells fromproliferating in vivo, for example cancer cells, autoreactive T cells,alloreactive T cells, HIV infected T cells, and the like (see King, etal., 2004, Cell 117:265-277). Accordingly, the compositions describedherein comprising T cells that have been cultured so as to delete atleast a substantial portion of unwanted cells can be infused into apatient. The infused cells then prevent homeostatic proliferation andtherefore, prevent unwanted cells from regenerating in vivo.

The administration of the subject pharmaceutical compositions may becarried out in any convenient manner, including by aerosol inhalation,injection, ingestion, transfusion, implantation or transplantation. Thecompositions of the present invention may be administered to a patientsubcutaneously, intradermally, intramuscularly, by intravenous (i.v.)injection, intratumorally, or intraperitoneally. Preferably, the T cellcompositions of the present invention are administered by i.v.injection. The compositions of activated T cells may be injecteddirectly into a tumor or lymph node.

In yet another embodiment, the pharmaceutical composition can bedelivered in a controlled release system. In one embodiment, a pump maybe used (see Langer, 1990, Science 249:1527-1533; Sefton 1987, CRC Crit.Ref. Biomed. Eng. 14:201; Buchwald et al., 1980; Surgery 88:507; Saudeket al., 1989, N. Engl. J. Med. 321:574). In another embodiment,polymeric materials can be used (see Medical Applications of ControlledRelease, 1974, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla.;Controlled Drug Bioavailability, Drug Product Design and Performance,1984, Smolen and Ball (eds.), Wiley, New York; Ranger and Peppas, 1983;J. Macromol. Sci. Rev. Macromol. Chem. 23:61; see also Levy et al.,1985, Science 228:190; During et al., 1989, Ann. Neurol. 25:351; Howardet al., 1989, J. Neurosurg. 71:105). In yet another embodiment, acontrolled release system can be placed in proximity of the therapeutictarget, thus requiring only a fraction of the systemic dose (see, e.g.,Medical Applications of Controlled Release, 1984, Langer and Wise(eds.), CRC Pres., Boca Raton, Fla., vol. 2, pp. 115-138).

The T cell and/or sensitizing composition compositions of the presentinvention may also be administered using any number of matrices.Matrices have been utilized for a number of years within the context oftissue engineering (see, e.g., Principles of Tissue Engineering (Lanza,Langer, and Chick (eds.)), 1997. The present invention utilizes suchmatrices within the novel context of acting as an artificial lymphoidorgan to support, maintain, or modulate the immune system, typicallythrough modulation of T cells. Accordingly, the present invention canutilize those matrix compositions and formulations which havedemonstrated utility in tissue engineering. Accordingly, the type ofmatrix that may be used in the compositions, devices and methods of theinvention is virtually limitless and may include both biological andsynthetic matrices. In one particular example, the compositions anddevices set forth by U.S. Pat. Nos. 5,980,889; 5,913,998; 5,902,745;5,843,069; 5,787,900; or 5,626,561 are utilized. Matrices comprisefeatures commonly associated with being biocompatible when administeredto a mammalian host. Matrices may be formed from both natural andsynthetic materials. The matrices may be non-biodegradable in instanceswhere it is desirable to leave permanent structures or removablestructures in the body of an animal, such as an implant; orbiodegradable. The matrices may take the form of sponges, implants,tubes, telfa pads, fibers, hollow fibers, lyophilized components, gels,powders, porous compositions, liposomes, cells, or nanoparticles. Inaddition, matrices can be designed to allow for sustained release ofseeded cells or produced cytokine or other active agent. In certainembodiments, the matrix of the present invention is flexible andelastic, and may be described as a semisolid scaffold that is permeableto substances such as inorganic salts, aqueous fluids and dissolvedgaseous agents including oxygen.

A matrix is used herein as an example of a biocompatible substance.However, the current invention is not limited to matrices and thus,wherever the term matrix or matrices appears these terms should be readto include devices and other substances which allow for cellularretention or cellular traversal, are biocompatible, and are capable ofallowing traversal of macromolecules either directly through thesubstance such that the substance itself is a semi-permeable membrane orused in conjunction with a particular semi-permeable substance.

All references referred to within the text are hereby incorporated byreference in their entirety. Moreover, all numerical ranges utilizedherein explicitly include all integer values within the range andselection of specific numerical values within the range is contemplateddepending on the particular use. Further, the following examples areoffered by way of illustration, and not by way of limitation.

EXAMPLE 1 Deletion of Antigen-Specific T Cells Following Restimulationwith CD3/CD28 XCELLERATE™ Beads

This example describes the elimination of antigen-specific T cells froma mixed population of cells by restimulation with anti CD3/CD28XCELLERATE™ beads (3×28 beads). The generation of XCELLERATED T cells™using the processes described herein is essentially as described in U.S.patent application Ser. No. 10/133,236.

Human PBMC were screened for HLA-A2 CMVpp65 positivity by flow cytometryusing HLA-A2 tetramers loaded with CMVpp65 peptide (HLA-A2-CMVpp65).Approximately 3% of the CD3+ CD8+T cells in the donor selected expressedTCR specific for HLA-A2-CMVpp65 (FIG. 1).

PBMC from the donor (donor 2) and control donor (donor 1) were activatedwith CMV antigen coated onto paramagnetic beads and by day 10 ofculture, many cells were shown by flow cytometric analysis to be CD25(IL-2R) positive, and all of the HLA-A2 CMVpp65+T cells expressed highlevels of CD25, indicating activation (FIG. 2, right panel).

At day 14 post-primary stimulation, cultures were then either leftunstimulated (FIG. 3, panels A1-A4) or were restimulated using theXCELLERATE™ process with 3×28 beads for 16 hours (FIG. 3, panels B1-B4).As shown in FIG. 3, CD25 is upregulated on restimulated cells (panelB2), but tetramer-positive (i.e., CMVpp65-Ag-specific) prestimulatedcells were deleted by the secondary strong stimulation provided by the3×28 beads (panels B3 and B4), while the other cells were unaffected.Similar results were observed when cells were attached to beads orassociated with cells attached to beads, magnetically selected andplaced back into culture prior to restimulation with the 3×28 beads. Inan additional study, the cells were restimulated for an additional 4days. Deletion of the tetramer-positive cells was still observed after 4additional days in the 3×28 restimulated cultures.

These results demonstrate that activated CMVpp65-antigen-specific Tcells that are restimulated with 3×28 beads are eliminated from thepopulation of cells, most likely through apoptosis.

EXAMPLE 2 Determination of Apoptosis

This example describes an illustrative assay for measuring apoptosis.

DNA Fragmentation Assay: Cells are lysed in 50 μl of lysis buffer (10 mMEDTA, 50 mM Tris pH 8, 0.5% sodium dodecyl sulfate, 0.5 mg/ml proteinaseK). RNAse A (0.5 mg/ml) is added and lysates are incubated for 1 hr. at37° C. Two phenol extraction (equal volumes) are performed, followed byone chloroform extraction. DNA is precipitated with two volumes ofice-cold ethanol and incubated at −80° C. for 1 hr. DNA is pelleted bycentrifugation at 14,000 rpm for 10 minutes at 4° C. Pellets areair-dried for 30 minutes, resuspended in 50 μl of Tris-EDTA pH 8. DNA iselectrophoresed in a 1.8% agarose gel in 1×TBE running buffer (0.05 MTris base, 0.05 M boric acid, 1 mM disodium EDTA), according to themethods of Preston, et al., Cancer Res., 1994, 54, 4214-4223.

EXAMPLE 3 Induction of Apoptosis in B-Cells by Coculture withXCELLERATED T Cells™

This example describes the deletion of leukemic B-cells in B-CLL patientsamples by co-culture with XCELLERATED T cells™.

XCELLERATED T cells™, generated essentially as described in U.S. patentapplication Ser. No. 10/133,236, were co-cultured with unmanipulatedautologous leukemic cells from B-CLL patients. Cell surface markers forCD54, CD80, CD95 (FAS) and CD86, and Annexin/PI (apoptosis) weremeasured at 24 and 48 hours by flow cytometry. XCELLERATED T cells™ wereshown to drive up expression of CD95 (FAS) on leukemic B cells (FIG. 4).After 48 hours of co-culture with day 12 XCELLERATED T cells™,autologous leukemic B cells show increased expression of CD95 andsensitivity to anti-FAS as measured by flow cytometry (FIG. 5). As shownin FIG. 5, addition of anti-FAS antibody to co-cultured T:B cells led toincreased apoptosis in the leukemic B-cells. In an additional study, itwas shown that T cells grow whereas leukemic B-cells are eliminatedduring the XCELLERATE™ process (FIG. 6).

In summary, XCELLERATED T cells™ upregulate important effector moleculeson leukemic B cells, induce functional FAS on leukemic B-cells, and candrive leukemic B-cells into apoptosis pathways. Leukemic B cells werevirtually undetectable by the end of the XCELLERATE™ process. Therefore,XCELLERATED T cells™ can be used as a sensitizing composition or apro-apoptotic composition for the elimination of leukemic B-cells from amixed population of cells.

EXAMPLE 4 Varying Bead:Cell Ratios can Selectively Expand or DeleteMemory CD8 T Cells

This example shows that the bead:cell ratio can have a profound effecton expansion of different populations of T cells. In particular, a highbead:cell ratio (3:1-10:1) tends to induce death in antigen-specific Tcells while a lower bead:cell ratio (1:1-1:10) leads to expansion ofantigen-specific T cells. Further, the data described below show thatlower bead:cell ratios lead to improved cell expansion in polyclonalcell populations as well. Thus, this example shows that lower bead:cellratios improve overall cell expansion. Further, this exampledemonstrates that at high bead:cell ratios, the beads described hereincan be used as pro-apoptotic compositions.

Cells were prepared and stimulated using the XCELLERATE I™ processessentially as described in U.S. patent application Ser. No. 10/187,467filed Jun. 28, 2002. Briefly, in this process, the XCELLERATED T cells™are manufactured from a peripheral blood mononuclear cell (PBMC)apheresis product. After collection from the patient at the clinicalsite, the PBMC apheresis are washed and cryopreserved. Cells were thenthawed, and placed in culture @37° C./5% CO₂ for 1 hour to allowmonocytes and other adherent cells to bind to the culture plate.Non-adherent cells were transferred to new culture plates forstimulation as follows. Following this monocyte-depletion step, a volumecontaining a total of 5×10⁸ CD3⁺ T cells is taken and set-up with1.5×10⁹ DYNABEADS® M-450 CD3/CD28 T to initiate the XCELLERATE™ process(approx. 3:1 beads to T cells). The mixture of cells and DYNABEADS®M-450 CD3/CD28 T are then incubated at 37° C., 5% CO₂ for approximately8 days to generate XCELLERATED T cells™ for a first infusion. Theremaining monocyte-depleted PBMC are cryopreserved until a second orfurther cell product expansion (approximately 21 days later) at whichtime they are thawed, washed and then a volume containing a total of5×10⁸ CD3⁺ T cells is taken and set-up with 1.5×10⁹ DYNABEADS® M-450CD3/CD28 T to initiate the XCELLERATE™ Process for a second infusion.During the incubation period of ≈8 days at 37° C., 5% CO₂, the CD3⁺ Tcells activate and expand. The anti-CD3 mAb used is BC3 (XR-CD3; FredHutchinson Cancer Research Center, Seattle, Wash.), and the anti-CD28mAb (B-T3, XR-CD28) is obtained from Diaclone, Besançon, France.

For the experiment described below, cultures containing cells for whichadherent cells had been removed then have beads added at bead:T cellratios as shown in Table 1. The beads used in this Example comprised theDYNABEADS® M-450 CD3/CD28 T with a 1:1 CD3:CD28 antibody ratio bound onthe beads. TABLE 1 Varying Bead:Cell Ratios can Selectively Expand orDelete Memory CD8 T cells Fold Increase Bead:Cell Ratio Polyclonal Tcells CMV Antigen-Specific T cells 10:1  149 0 5:1 294 0 3:1 346 1.4 1:1562 20.6 1:5 113 53  1:10 79 45.8

The results summarized in Table 1 and shown graphically in FIG. 7demonstrate that antigen-specific T cells can be selectively deleted byusing high bead:cell ratios and expanded using low bead:cell ratios.Similar results were observed with EBV-specific CD8⁺ T cells andinfluenza-specific CD8⁺ T cells and CD4⁺ T cells (not shown). Withoutbeing bound by theory, it is thought that the antigen-specific T cellsare sensitized to further stimulation. Stimulation with high bead:cellratios provides a high concentration of stimulating antibody, leading toover-stimulation of antigen-specific T cells, causing them to die,either by apoptosis or other mechanisms. Thus, in this regard, the beadsare functioning as a pro-apoptotic composition. Using lower bead:cellratios provides a stimulation signal to antigen-specific T cells thatdoes not over-stimulate, but rather induces rapid proliferation of thesecells. An increase in proliferation is also observed in the polyclonalpopulation of T cells using lower bead:cell ratios. In particular, theresults indicate that a bead:cell ratio of 1:1 is optimal for polyclonalT cell expansion.

Therefore, in this Example, evidence is provided to support the use ofdiffering bead:cell ratios depending on the outcome desired. Forexpansion of antigen-specific T cells, a lower bead:cell ratio ispreferable. If deletion of antigen-specific T cells is the desiredoutcome, a higher bead:cell ratio is preferable.

EXAMPLE 5 Deletion of Allo-Reactive T Cells Following Restimulation withCD3/CD28 XCELLERATE™ Beads

This example describes the deletion of allo-reactive T cells followingrestimulation with CD3/CD28 XCELLERATE™ beads.

PBMC were stimulated for 3 days with either allogeneic PBMC or the JYB-lymphoblastoid allogeneic cell line. On day 3, the allogeneic PBMC- orJY-stimulated PBMC were then cultured with CD3/CD28 beads using theXCELLERATE™ process essentially as described in U.S. patent applicationSer. No. 10/350,305, with and without 30 minute positive selection withCD3/CD28 beads. Following the XCELLERATE™ process, the cells were thenrestimulated with either allogeneic PBMC or JY allogeneic antigen andCD25 up-regulation was measured. Restimulation with allogeneic cellsfollowing the XCELLERATE™ process did not lead to upregulation of CD25expression (measured using flow cytometric analysis), indicating thatthe allo-reactive cells had been deleted. In particular, positiveselection of JY stimulated CD8+ T cells during the XCELLERATE™ processsignificantly decreased allo-reactivity. However, the T cells remainedcompetent to respond to irrelevant antigens in XCELLERATED™ cultures asdemonstrated by 3rd party allogeneic PBMC and JY responses (e.g.,restimulation of JY-stimulated culture with allogeneic PBMC orrestimulation of allo-PBMC-stimulated culture with JY).

Thus, these results show that activated allo-reactive T cells aredeleted by restimulation with CD3/CD28 beads while the remainingpolyclonal T cells can be expanded exponentially for use in any numberof immunotherapeutic applications.

EXAMPLE 6 Restoration of T Cell Repertoire in Patients with AutoimmuneDisease

T cells from patients with autoimmune disease were expanded using theXCELLERATE™ process and T cell repertoire observed by spectratypeanalysis.

Samples from patients with systemic lupus erythematosus, rheumatoidarthritis, scleroderma, Crohn's disease, and psoriatic arthritis wereanalyzed. Total T cell expansion in these patients (n=9) using theXCELLERATE™ process was similar to that observed in normal donors (asseen in FIG. 7) using bead:T cell ratios from 1:5 to 5:1. Further studyof T cells from patients with rheumatoid arthritis, psoriatic arthritis,and Crohn's disease using spectratype analysis showed that a healthy Tcell repertoire is restored following the XCELLERATE™ process (5:1bead:T cell ratio) (Representative sample from a patient with rheumatoidarthritis is shown in FIG. 8. Similar results were observed in a patientwith psoriatic arthritis and a Crohn's disease patient (not shown)).Further analysis showed that XCELLERATED™ T cells from these patientsexhibit a Th1 phenotype (see FIG. 9).

In summary, in autoimmune disease patients, the XCELLERATE™ process canbe used to expand T cells more than one thousand fold, restore a broad Tcell repertoire and generate a Th 1-type T cell population.

EXAMPLE 7 High Bead:T Cell Ratio Deletes Autoreactive CD8+ T Cells in aMouse Diabetes Model

In a related experiment, the XCELLERATE™ process (5:1 bead:T cell ratio)was used to expand T cells in a mouse diabetes model. Cells wereexpanded using the XCELLERATE™ process and further analyzed using anislet-specific MHC Class I tetramer. The results shown in FIG. 10demonstrate that the autoreactive CD8+ T cells were deleted.

In summary, using a pre-clinical mouse diabetes model, the XCELLERATE™process eliminated autoreactive CD8+ T cells.

All of the above U.S. patents, U.S. patent application publications,U.S. patent applications, foreign patents, foreign patent applicationsand non-patent publications referred to in this specification and/orlisted in the Application Data Sheet, are incorporated herein byreference, in their entirety.

From the foregoing it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

1. A method for eliminating at least a substantial portion of a clonal Tcell subpopulation from a mixed population of T cells from anindividual, comprising, exposing a population of cells, wherein at leasta portion thereof comprises T cells, to one or more pro-apoptotic orgrowth inhibiting compositions wherein said exposure induces apoptosisor growth inhibition in at least a substantial portion of at least oneclonal T cell population present in the mixed population of T cells;thereby eliminating at least a substantial portion of said clonal T cellpopulation from the mixed population of T cells.
 2. The method of claim1 further comprising expanding the remaining mixed population of Tcells.
 3. The method of claim 2 wherein the remaining mixed populationof cells is expanded by exposing the remaining mixed population of cellsto a surface wherein the surface has attached thereto one or more agentsthat ligate a cell surface moiety of at least a portion of the remainingT cells and stimulates said remaining T cells.
 4. The method of claim 3,wherein said surface has attached thereto a first agent that ligates afirst T cell surface moiety of a T cell, and the same or a secondsurface has attached thereto a second agent that ligates a second moietyof said T cell, wherein said ligation by the first and second agentinduces proliferation of said T cell.
 5. A population of T cellsgenerated according to the method of any one of claims 1-3.
 6. Themethod of claim 1 wherein the pro-apoptotic or growth inhibitingcomposition comprises an autoantigen.
 7. The method of claim 6, whereinthe autoantigen is selected from the group consisting of myelin basicprotein (MBP), MBP 84-102, MBP 143-168, pancreatic islet cell antigens,collagen, thyroid antigens, Scl-70, nucleic acid, acetylcholinereceptor, S Antigen, and type II collagen.
 8. The method of claim 1wherein the pro-apoptotic composition comprises allogeneic or xenogeneiccells.
 9. The method of claim 1 wherein said population of cells,wherein at least a portion thereof comprises T cells, is exposed to oneor more pro-apoptotic compositions in vivo.
 10. The method of claim 1wherein said population of cells, wherein at least a portion thereofcomprises T cells, is exposed to one or more pro-apoptotic compositionsex vivo.
 11. The method of claim 3 wherein the exposure of said cells tosaid surface is for a time sufficient to increase polyclonality.
 12. Themethod of claim 11 wherein the increase comprises a shift from mono tooligoclonality or to polyclonality of the T cell population as measuredby a Vβ, Vα, Vγ, or Vδ spectratype profile of at least one Vβ, Vα, Vγ,or Vδ family gene.
 13. A population of T cells generated according tothe method of claim 6 or
 11. 14. A method for treating autoimmunedisease in a patient comprising administering to the patient thepopulation of T cells of claim
 13. 15. The method of claim 14 whereinthe patient has been treated with an immunoablative agent prior toadministering the population of T cells of claim
 10. 16. The method ofclaim 15 wherein the immunoablative agent is selected from the groupconsisting of campath, anti-CD3 antibodies, cyclophosphamide,fludarabine, cyclosporine, FK506, mycophenolic acid, steroids, FR901228,and irradiation.
 17. The method of claim 14 wherein the patient has beentreated with a T cell ablative therapy prior to administering thepopulation of T cells of claim
 10. 18. The method of claim 1 wherein thepro-apoptotic or growth inhibiting composition comprises one or morecompositions selected from the group consisting of, anti-CD3 antibody,anti-CD2 antibody, anti-CD20 antibody, target antigen, MHC-peptidetetramers or dimers, Fas ligand, anti-Fas antibody, IL-2, IL-4, TRAIL,rolipram, doxorubicin, chlorambucil, fludarabine, cyclophosphamide,azathioprine, methotrexate, cyclosporine, mycophenolate, FK506,inhibitors of bcl-2, topoisomerase inhibitors, interleukin-1β convertingenzyme (ICE)-binding agents, Shigella IpaB protein, staurosporine,ultraviolet irradiation, gamma irradiation, tumor necrosis factor,target antigens nucleic acid molecules, proteins or peptides, andnon-protein or non-polynucleotide compounds.
 19. The method of claim 3,wherein at least one agent is an antibody or an antibody fragment. 20.The method of claim 3, wherein the first agent is an antibody or afragment thereof, and the second agent is an antibody or a fragmentthereof.
 21. The method of claim 3, wherein the first and the secondagents are different antibodies.
 22. The method of claim 3, wherein thefirst agent is an anti-CD3 antibody, an anti-CD2 antibody, or anantibody fragment of an anti-CD3 or anti-CD2 antibody.
 23. The method ofclaim 3, wherein the second agent is an anti-CD28 antibody or antibodyfragment thereof.
 24. The method of claim 3, wherein the first agent isan anti-CD3 antibody and the second agent is an anti-CD28 antibody. 25.A method for eliminating at least a substantial portion of a clonal Tcell subpopulation from a mixed population of T cells from anindividual, comprising, (a) exposing a population of cells wherein atleast a portion thereof comprises T cells to one or more compositionsthat sensitize at least a portion of the T cells to further activationor stimulation, (b) exposing the population of cells to a surfacewherein the surface has attached thereto one or more agents that ligatea cell surface moiety of at least a portion of the sensitized T cellsand stimulates said sensitized T cells, wherein the exposure of saidsensitized T cells to said surface is for a time sufficient to induceapoptosis of said sensitized T cells; thereby eliminating saidsensitized T cells from the population.
 26. The method of claim 25wherein step (b) further comprises exposing said population of cells tosaid surface for a time sufficient to stimulate at least a portion ofthe remaining T cells and wherein said at least a portion of theremaining cells proliferates.
 27. The method of claim 25, wherein saidsurface has attached thereto a first agent that ligates a first T cellsurface moiety of a T cell; and the same or a second surface hasattached thereto a second agent that ligates a second moiety of said Tcell, wherein said ligation by the first and second agent inducesproliferation of said T cell.
 28. The method of claim 26 wherein theexposure of said cells to said surface is for a time sufficient toincrease polyclonality.
 29. The method of claim 28 wherein the increasecomprises a shift from mono to oligoclonality or to polyclonality of theT cell population as measured by a Vβ, Vα, Vγ, or Vδ spectratype profileof at least one Vβ, Vα, Vγ, or Vδ family gene.
 30. A population of Tcells generated according to the method of claim
 28. 31. The method ofclaim 25 wherein the individual requires a hematopoietic stem celltransplant.
 32. The method of claim 31, wherein the composition thatsensitizes comprises recipient PBMCs that have been treated such thatthey are unable to continue dividing and the population of cellscomprises donor T cells.
 33. A population of T cells generated accordingto the method of claim
 32. 34. A method for reducing the risk of, or theseverity of, an adverse GVHD effect in a patient who is undergoing ahematopoietic stem cell transplant, comprising administering to saidpatient the population of T cells according to claim 30 or
 33. 35. Themethod of claim 25 wherein the individual requires an organ transplant.36. The method of claim 35 wherein the composition that sensitizescomprises donor cells that have been treated such that they are unableto divide and the population of cells comprises recipient T cells. 37.The method of claim 36 wherein the exposure of said cells to saidsurface is for a time sufficient to increase polyclonality.
 38. Themethod of claim 37 wherein the increase comprises a shift from mono tooligoclonality or to polyclonality of the T cell population as measuredby a Vβ, Vα, Vγ, or Vδ spectratype profile of at least one Vβ, Vα, Vγ,or Vδ family gene.
 39. A population of T cells generated according tothe method of claim 36 or
 37. 40. A method for reducing the risk oforgan rejection in a patient who is receiving an organ transplant,comprising administering to the patient the population of T cells ofclaim
 39. 41. The method of claim 40 wherein the patient has beentreated with a T cell ablative therapy prior to administration of thepopulation of T cells of claim
 36. 42. The method of claim 25 whereinthe composition that sensitizes comprises an autoantigen.
 43. The methodof claim 42, wherein the autoantigen is selected from the groupconsisting of myelin basic protein (MBP), MBP 84-102, MBP 143-168,Scl-70, pancreatic islet cell antigens, S Antigen; and type II collagen.44. The method of claim 43 wherein the exposure of said cells to saidsurface is for a time sufficient to increase polyclonality.
 45. Themethod of claim 44 wherein the increase comprises a shift from mono tooligoclonality or to polyclonality of the T cell population as measuredby a Vβ, Vα, Vγ, or Vδ spectratype profile of at least one Vβ, Vα, Vγ,or Vδ family gene.
 46. A population of T cells generated according tothe method of claim
 42. 47. A method for treating autoimmune disease ina patient comprising administering to the patient the population of Tcells of claim
 46. 48. The method of claim 47 wherein the patient hasbeen treated with a T cell ablative therapy prior to administering thepopulation of T cells of claim
 46. 49. The method of claim 26, whereinat least one agent is an antibody or an antibody fragment.
 50. Themethod of claim 26, wherein the first agent is an antibody or a fragmentthereof, and the second agent is an antibody or a fragment thereof. 51.The method of claim 50, wherein the first and the second agents aredifferent antibodies.
 52. The method of claim 26, wherein the firstagent is an anti-CD3 antibody, an anti-CD2 antibody, or an antibodyfragment of an anti-CD3 or anti-CD2 antibody.
 53. The method of claim26, wherein the second agent is an anti-CD28 antibody or antibodyfragment thereof.
 54. The method of claim 26, wherein the first agent isan anti-CD3 antibody and the second agent is an anti-CD28 antibody.55-61. (canceled)
 62. A method for activating and expanding a populationof T cells by cell surface moiety ligation, comprising: contacting apopulation of cells, wherein at least a portion thereof comprises Tcells, with a surface, wherein said surface has attached thereto one ormore agents that ligate a cell surface moiety of at least a portion ofthe T cells and stimulates said T cells, wherein said surface is presentat a ratio of said surface to said cells such that at least asubstantial portion of at least one population of antigen-specific Tcells is deleted after about 8 days of culture.
 63. The method of claim62 wherein said ratio is from about 10:1 to about 5:1.
 64. The method ofclaim 62 wherein said ratio is about 5:1.
 65. The method of claim 62wherein said ratio is about 10:1.
 66. The method of claim 1 furthercomprising expanding the mixed population of T cells, comprising,exposing the remaining mixed population of T cells to the pro-apoptoticcomposition, wherein said exposure induces proliferation in the mixedpopulation of T cells.
 67. The method of claim 66 wherein saidpro-apoptotic composition comprises anti-CD3 and anti-CD28 antibodiesco-immobilized on a bead.
 68. A method for treating a patient afflictedwith an autoimmune disease comprising: (a) contacting a population ofcells from the patient, wherein at least a portion thereof comprises Tcells, with a surface, wherein said surface has attached thereto one ormore agents that ligate a cell surface moiety of at least a portion ofthe T cells and stimulates said T cells, wherein said surface is presentat a ratio of said surface to said cells such that at least asubstantial portion of at least one population of antigen-specific Tcells is deleted after about 8 days of culture; and (b) administering tothe patient an effective amount of T cells from (a) such that in vivohomeostatic proliferation is inhibited; thereby treating autoimmunedisease.
 69. The method of claim 68 wherein said ratio is from about10:1 to about 5:1.
 70. The method of claim 68 wherein said ratio isabout 5:1.
 71. The method of claim 68 wherein said ratio is about 10:1.72. A method for treating a patient afflicted with an autoimmune diseasecomprising: (a) contacting a population of cells from the patient,wherein at least a portion thereof comprises T cells, with a surface,wherein said surface has attached thereto one or more agents that ligatea cell surface moiety of at least a portion of the T cells andstimulates said T cells; (b) administering to the patient the T cells of(a) at a dose such that homeostatic proliferation of endogenous T cellsis inhibited; thereby treating autoimmune disease.
 73. A method fortreating a patient infected with HIV comprising: (a) contacting apopulation of cells from the patient, wherein at least a portion thereofcomprises T cells, with a surface, wherein said surface has attachedthereto one or more agents that ligate a cell surface moiety of at leasta portion of the T cells and stimulates said T cells, wherein saidsurface is present at a ratio of said surface to said cells such that atleast a substantial portion of HIV-infected T cells is deleted afterabout 8 days of culture; and (b) administering to the patient aneffective amount of T cells from (a) such that in vivo homeostaticproliferation is inhibited; thereby treating the patient infected withHIV.